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Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization
Abstract
SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating the conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI's oligomerization and allosteric behavior.
... There are three primary sequences, following the pattern CR|CCGGYG (| denotes cleavage site, Y = C or T, R = A or G), and fourteen secondary sequences following the patterns DRCCGGYG (where D = A, G, or T) or CCCCGGYG, giving a total of seventeen sequences cleaved by SgrAI in the activated state [43]. Through a series of biochemical, biophysical, and structural analyses, the activated state of SgrAI was demonstrated to be filamentous [25,44,45], formed by the assembly of DNA bound SgrAI dimers (or DBDs, ∼100 kDa each) into a left-handed helix, which is held together largely by interactions between SgrAI enzymes, with the bound DNA looping in and out of the filament [45]. Only primary sequences will stimulate filament formation by SgrAI, whereas secondary sequences will not. ...
... There are three primary sequences, following the pattern CR|CCGGYG (| denotes cleavage site, Y = C or T, R = A or G), and fourteen secondary sequences following the patterns DRCCGGYG (where D = A, G, or T) or CCCCGGYG, giving a total of seventeen sequences cleaved by SgrAI in the activated state [43]. Through a series of biochemical, biophysical, and structural analyses, the activated state of SgrAI was demonstrated to be filamentous [25,44,45], formed by the assembly of DNA bound SgrAI dimers (or DBDs, ∼100 kDa each) into a left-handed helix, which is held together largely by interactions between SgrAI enzymes, with the bound DNA looping in and out of the filament [45]. Only primary sequences will stimulate filament formation by SgrAI, whereas secondary sequences will not. ...
... However, SgrAI bound to secondary sequences will join into filaments formed by SgrAI bound to primary sequences. This filament could, in principle, extend infinitely, but in both experimental and biological settings is expected to be limited to sizes of twenty DBDs or less [45]. Detailed studies on the structure, kinetic mechanism, and biological role of SgrAI filamentation have shed light on how filamentation regulates this enzyme's behavior. ...
Filament formation by metabolic, biosynthetic, and other enzymes has recently come into focus as a mechanism to fine-tune enzyme activity in the cell. Filamentation is key to the function of SgrAI, a sequence-specific DNA endonuclease that has served as a model system to provide some of the deepest insights into the biophysical characteristics of filamentation and its functional consequences. Structure-function analyses reveal that, in the filamentous state, SgrAI stabilizes an activated enzyme conformation that leads to accelerated DNA cleavage activity and expanded DNA sequence specificity. The latter is thought to be mediated by sequence-specific DNA structure, protein–DNA interactions, and a disorder-to-order transition in the protein, which collectively affect the relative stabilities of the inactive, non-filamentous conformation and the active, filamentous conformation of SgrAI bound to DNA. Full global kinetic modeling of the DNA cleavage pathway reveals a slow, rate-limiting, second-order association rate constant for filament assembly, and simulations of in vivo activity predict that filamentation is superior to non-filamenting mechanisms in ensuring rapid activation and sequestration of SgrAI's DNA cleavage activity on phage DNA and away from the host chromosome. In vivo studies demonstrate the critical requirement for accelerated DNA cleavage by SgrAI in its biological role to safeguard the bacterial host. Collectively, these data have advanced our understanding of how filamentation can regulate enzyme structure and function, while the experimental strategies used for SgrAI can be applied to other enzymatic systems to identify novel functional roles for filamentation.
... Our particular interest in this phenomenon originated with our studies of SgrAI, a type II restriction endonuclease with unusual allosteric behavior, where binding to one type of DNA sequence results in activation of the enzyme to cleave 14 additional DNA sequences (Bitinaite and Schildkraut 2002). Our investigation into the mechanism responsible for this behavior led to the discovery of filament formation by SgrAI when bound to the activating DNA (which is also a substrate for cleavage of SgrAI known as primary site sequences) Lyumkis et al. 2013;Ma et al. 2013). The filamentous form recruits additional copies of SgrAI bound to the second type of DNA sequence (secondary sites) Shah et al. 2015). ...
... SgrAI forms filaments when bound to primary site DNA sequences Lyumkis et al. 2013;Ma et al. 2013), and its structure has been characterized to 3.5 Å resolution by helical reconstruction and cryo-electron microscopy (Polley et al. 2019). The filament is a left-handed helix with approximately 4 copies of the SgrAI/DNA complex per turn (Fig. 19c). ...
... Some other unique features of the SgrAI system are also found, including filament formation being stimulated by both substrate and product (of the primary site sequence), participation of the substrate (and product) in stabilization of the filament via direct interactions with neighboring protomers, and the requirement to dissociate from the filament in order to release product Lyumkis et al. 2013;Shah et al. 2015;Park et al. 2018a;Park et al. 2018b;Barahona et al. 2019). We found that the dynamics of SgrAI enzymes into and out of the filament is sufficiently fast to prevent trapping of the product, despite this requirement. ...
We present some of the diversity of functional, biologically relevant, enzymatic filament structures. The recent advances in CryoEM and associated structural computational methods, coupled with whole genome screens, have helped identify and recognize these oligomers as important in different areas such as regulation, activation, selectivity, and substrate channeling.
... We study type II restriction endonuclease SgrAI to better understand the molecular mechanism of enzyme filamentation in the control of enzyme activity and to define the biological advantages filamentation provides over other forms of enzyme regulation. Our discovery that SgrAI forms filaments was completely unexpected, and our work has since shown that filamentation leads to altered enzymatic properties in both the rate of DNA cleavage by SgrAI and in its substrate specificity (i.e., the sequences of DNA cleaved by the enzyme) (12)(13)(14). Under basal conditions, SgrAI is a homodimeric and sequence-specific restriction endonuclease of 74 kDa with two active sites, one in each subunit. ...
... Data from ion mobility native mass spectrometry not only confirmed the heterogeneous nature of SgrAI-DNA assemblies, but also indicated that they exhibited a regular repeating structure, which is suggestive of a filament (13). EM was instrumental in demonstrating the filamentous nature of SgrAI bound to its primary site sequence and revealed that DNA-bound SgrAI assembles into filaments in a run-on manner, characterized by the addition of individual DBDs at either end (14). Additional biophysical and DNA cleavage studies indicated that SgrAI bound to secondary sequences cannot alone induce filamentation but will assemble into filaments formed by SgrAI bound to primary site DNA (12). ...
... Additional biophysical and DNA cleavage studies indicated that SgrAI bound to secondary sequences cannot alone induce filamentation but will assemble into filaments formed by SgrAI bound to primary site DNA (12). Since DNA cleavage activity is greatly accelerated in the filamentous form, we proposed that interactions between DBDs within the filament stabilize an activated conformation of the enzyme that is distinct from that exhibited in the nonfilamentous state (12,14,16). Cleavage of secondary sequences under activating conditions is explained by our model in that DBDs containing secondary sequences will be drawn into filaments composed of DBDs containing primary sequences, which activates the filamentous assembly to rapidly cleave bound secondary site DNAs (12,17). ...
Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes – the acceleration of DNA cleavage and the expansion of sequence specificity – are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca²⁺ resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme/DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein-DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.
... II RE SgrAI from Streptomyces griseus led us to propose a new mechanism of enzyme regulation involving filament formation (7,8). ...
... At approximately the same time, large-scale screens for protein localization using fluorescence microscopy showed unexpectedly that many enzymes formed filaments in response to particular metabolic conditions or other stimuli in cells (11,(13)(14)(15). The term "run-on oligomer" (ROO) filament is used here to describe an assembly of an enzyme into a filament by the successive addition of enzymes at either end, and which, in principle, could extend indefinitely (8,16). ROO filament formation by SgrAI was first proposed in 2010 based on behavior in analytical ultracentrifugation and native gels (7) and subsequently using ion-mobility mass spectrometry (17). ...
... ROO filament formation by SgrAI was first proposed in 2010 based on behavior in analytical ultracentrifugation and native gels (7) and subsequently using ion-mobility mass spectrometry (17). The enzymatic activity of SgrAI was found to be activated in the ROO and to possess an altered (expanded) DNA sequence specificity (7,8). The three-dimensional cryoelectron microscopy (cryo-EM) structure of the ROO filament formed by the assembly of SgrAI/DNA complexes shows a left-handed helical arrangement with approximately four DNA-bound dimers of SgrAI per turn (Fig. 1A to D show different views of an individual SgrAI/DNA complex) (8). ...
... Our particular interest in this phenomenon originated with our studies of SgrAI, a type II restriction endonuclease with unusual allosteric behavior, where binding to one type of DNA sequence results in activation of the enzyme to cleave 14 additional DNA sequences (Bitinaite and Schildkraut 2002). Our investigation into the mechanism responsible for this behavior led to the discovery of filament formation by SgrAI when bound to the activating DNA (which is also a substrate for cleavage of SgrAI known as primary site sequences) Lyumkis et al. 2013;Ma et al. 2013). The filamentous form recruits additional copies of SgrAI bound to the second type of DNA sequence (secondary sites) Shah et al. 2015). ...
... SgrAI forms filaments when bound to primary site DNA sequences Lyumkis et al. 2013;Ma et al. 2013), and its structure has been characterized to 3.5 Å resolution by helical reconstruction and cryo-electron microscopy (Polley et al. 2019). The filament is a left-handed helix with approximately 4 copies of the SgrAI/DNA complex per turn (Fig. 19c). ...
... Some other unique features of the SgrAI system are also found, including filament formation being stimulated by both substrate and product (of the primary site sequence), participation of the substrate (and product) in stabilization of the filament via direct interactions with neighboring protomers, and the requirement to dissociate from the filament in order to release product Lyumkis et al. 2013;Shah et al. 2015;Park et al. 2018a;Park et al. 2018b;Barahona et al. 2019). We found that the dynamics of SgrAI enzymes into and out of the filament is sufficiently fast to prevent trapping of the product, despite this requirement. ...
Filament formation by non-cytoskeletal enzymes has been known for decades, yet only relatively recently has its wide-spread role in enzyme regulation and biology come to be appreciated. This comprehensive review summarizes what is known for each enzyme confirmed to form filamentous structures in vitro, and for the many that are known only to form large self-assemblies within cells. For some enzymes, studies describing both the in vitro filamentous structures and cellular self-assembly formation are also known and described. Special attention is paid to the detailed structures of each type of enzyme filament, as well as the roles the structures play in enzyme regulation and in biology. Where it is known or hypothesized, the advantages conferred by enzyme filamentation are reviewed. Finally, the similarities, differences, and comparison to the SgrAI endonuclease system are also highlighted.
... II RE SgrAI from Streptomyces griseus led us to propose a new mechanism of enzyme regulation involving filament formation (7,8). ...
... At approximately the same time, large-scale screens for protein localization using fluorescence microscopy showed unexpectedly that many enzymes formed filaments in response to particular metabolic conditions or other stimuli in cells (11,(13)(14)(15). The term "run-on oligomer" (ROO) filament is used here to describe an assembly of an enzyme into a filament by the successive addition of enzymes at either end, and which, in principle, could extend indefinitely (8,16). ROO filament formation by SgrAI was first proposed in 2010 based on behavior in analytical ultracentrifugation and native gels (7) and subsequently using ion-mobility mass spectrometry (17). ...
... ROO filament formation by SgrAI was first proposed in 2010 based on behavior in analytical ultracentrifugation and native gels (7) and subsequently using ion-mobility mass spectrometry (17). The enzymatic activity of SgrAI was found to be activated in the ROO and to possess an altered (expanded) DNA sequence specificity (7,8). The three-dimensional cryoelectron microscopy (cryo-EM) structure of the ROO filament formed by the assembly of SgrAI/DNA complexes shows a left-handed helical arrangement with approximately four DNA-bound dimers of SgrAI per turn (Fig. 1A to D show different views of an individual SgrAI/DNA complex) (8). ...
Herein we investigate an unusual anti-viral mechanism developed in the bacterium Streptomyces griseus . SgrAI is a type II restriction endonuclease which forms run-on oligomer filaments when activated, and which possesses both accelerated DNA cleavage activity and expanded DNA sequence specificity. Mutations disrupting the run-on oligomer filament eliminate the robust anti-phage activity of wild type SgrAI, and the observation that even relatively modest disruptions completely abolish this anti-viral activity shows that the greater speed imparted by the run-on oligomer filament mechanism is critical to its biological function. Simulations of DNA cleavage by SgrAI uncover the origins of the kinetic advantage of this newly described mechanism of enzyme regulation over more conventional mechanisms, as well as the origin of the sequestering effect responsible for the protection of the host genome against the damaging DNA cleavage activity of activated SgrAI.
IMPORTANCE This work is motivated by the interest in understanding the characteristics and advantages of a relatively newly discovered enzyme mechanism involving filament formation. SgrAI is an enzyme responsible for protecting against viral infections in its host bacterium, and was one of the first such enzymes shown to utilize such a mechanism. In this work, filament formation by SgrAI is disrupted and the effects on the speed of the purified enzyme as well as its function in cells are measured. It was found that even small disruptions, which weaken but do not destroy filament formation, eliminate the ability of SgrAI to protect cells from viral infection, its normal biological function. Simulations of enzyme activity were also performed and show how filament formation can greatly speed up an enzyme’s activation compared to other known mechanisms, as well as better localize its action to molecules of interest such as invading phage DNA.
... Based on its biochemical activities, SgrAI, a nuclease from S. griseus, is postulated to be activated by binding to particular DNA sequences (primary sites) on invading phage DNA, simultaneously expanding its DNA sequence cleavage specificity and forming filaments of run-on oligomers (ROO 1 ). These filaments may act to protect the host DNA from its resulting off-target cleavage activity and to confer kinetic advantage in rapid DNA cleavage (2)(3)(4). Only recently is there a growing appreciation for the widespread nature and unique attributes of enzyme mechanisms involving filament formation (5)(6)(7)(8)(9)(10)(11)(12)(13). ...
... described previously, and dysfunction in the control of such pathways is implicated in human diseases including cancer, diabetes, and developmental problems (7,8). Being a relatively newly described enzyme mechanism (4,(14)(15)(16)(17) several fundamental questions concerning the role of the filament in biological function and enzyme activity remain to be answered, such as filament growth mechanisms, cooperativity, sequestration of activity, and advantages over non-ROO filament mechanisms. Further, potential limitations on enzyme turnover due to the requirement for filament assembly prior to enzyme activation, and/or in potentially trapping products of the reaction within the filament, have yet to be addressed. ...
... SgrAI is a sequence specific DNA enzyme and a type II restriction endonuclease with unusual allosteric properties and has been shown to form filaments we call ROO, for run-on oligomer, to describe the simple and symmetric nature of the assembly that can extend, in principle, indefinitely in either direction (3,4). The DNA cleavage activity of SgrAI is activated in the ROO filament by over 200-fold, and its DNA sequence specificity is also altered allowing cleavage of an additional class of DNA sequences termed secondary sites (3,4,18). ...
Filament or run-on oligomer formation by metabolic enzymes is now recognized as a widespread phenomenon having potentially unique enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA cleavage activity and altered DNA sequence specificity. However, the mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of enzyme activity, and advantages over other filament mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a non-cooperative growth mode of the run-on oligomer. These models and methods are extended in Part 2 to include the full DNA cleavage pathway, and address specific questions related to the run-on oligomer mechanism including the sequestration of DNA cleavage activity and trapping of products.
... Structural studies of SgrAI in its low activity form show a canonical dimeric RE fold enzyme bound to one recognition site (primary or secondary) in duplex DNA (14,15). These SgrAI/DNA complexes are then the building blocks for the high activity state of SgrAI which forms a left-handed helix with approximately four SgrAI/DNA complexes per turn which we call a run-on oligomer or ROO filament (Fig. 1A) (16). The SgrAI/DNA complexes associate using protein-protein and protein-DNA interactions between neighboring SgrAI/DNA complexes (Fig. 1A), and the DNA (when contiguous sites are bound by SgrAI) is predicted to weave in and out of the filament (17). ...
... The SgrAI/DNA complexes associate using protein-protein and protein-DNA interactions between neighboring SgrAI/DNA complexes (Fig. 1A), and the DNA (when contiguous sites are bound by SgrAI) is predicted to weave in and out of the filament (17). The ROO filament can also theoretically extended indefinitely from either end, and ROO filaments of 30 or more SgrAI/DNA complexes have been visualized via electron microscopy (16). The conformation of SgrAI is altered in the ROO filamentous activated state compared to the unoligomerized low activity state, as expected for an allosteric enzyme (16). ...
... The ROO filament can also theoretically extended indefinitely from either end, and ROO filaments of 30 or more SgrAI/DNA complexes have been visualized via electron microscopy (16). The conformation of SgrAI is altered in the ROO filamentous activated state compared to the unoligomerized low activity state, as expected for an allosteric enzyme (16). ...
Filament or run-on oligomer formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms run-on oligomeric filaments with activated DNA cleavage activity and altered DNA sequence specificity. In this two-part work, we measure individual steps in the run-on oligomer filament mechanism to address specific questions of cooperativity, trapping, filament growth mechanisms, and sequestration of activity using fluorophore labeled DNA, kinetic FRET measurements, and reaction modeling with global data fitting. The final models and rate constants show that the assembly step involving association of SgrAI/DNA complexes into the run-on oligomer filament is relatively slow (three to four orders of magnitude slower than diffusion limited) and rate limiting at low to moderate concentrations of SgrAI/DNA. The disassembly step involving dissociation of complexes of SgrAI/DNA from each other in the run-on oligomer filament is the next slowest step, but is fast enough to limit the residence time of any one copy of SgrAI or DNA within the dynamic filament. Further, the rate constant for DNA cleavage is found to be four orders of magnitude faster in the run-on oligomer filament than in isolated SgrAI/DNA complexes, and faster than dissociation of SgrAI/DNA complexes from the run-on oligomer filament, making the reaction efficient in that each association into the filament likely leads to DNA cleavage before filament dissociation.
... Type II restriction endonucleases (REases) belong to four different nuclease families: PD-(D/E)XK, PLD, HNH and GYI-IYG (1,2). PD-(D/E)XK family REases which recognize palindromic DNA sequences assemble into different oligomeric structures to generate double strand breaks in DNA (2,3). Orthodox Type IIP REases are arranged as dimers and each monomer contains an active site that acts on one DNA strand within a symmetrical target site. ...
... Tetrameric REases require binding to two target sites simultaneously and cleave four phosphodiester bonds in a concerted manner. Intermediate variants, exemplified by Ecl18kI, BsaWI and SgrAI, exist as dimers in the apo form, but cleave DNA as tetramers (Ecl18kI and BsaWI) or 'runon' oligomers (SgrAI) (3,5,6). Monomeric Type II restriction enzymes interact with their palindromic (MspI and HinP1I) or pseudo-palindromic (BcnI and MvaI) sites as monomers: a single protein subunit makes contacts with both parts of the palindromic target site (7)(8)(9)(10). ...
... Some REases, as exemplified by Cfr10I, Bse634I and NgoMIV, are homotetramers, which bind and cleave two target sites simultaneously (45,46,55). Other restriction enzymes like Ecl18kI, SgrAI and BsaWI are dimers in the apo form and make tetramers or higher order oligomers when bound to DNA (3,5,6). DNA cleavage mechanism of AgeI is most similar to that of the Type IIS enzyme FokI. ...
Although all Type II restriction endonucleases catalyze phosphodiester bond hydrolysis within or close to their DNA target sites, they form different oligomeric assemblies ranging from monomers, dimers, tetramers to higher order oligomers to generate a double strand break in DNA. Type IIP restriction endonuclease AgeI recognizes a palindromic sequence 5'-A/CCGGT-3' and cuts it ('/' denotes the cleavage site) producing staggered DNA ends. Here, we present crystal structures of AgeI in apo and DNA-bound forms. The structure of AgeI is similar to the restriction enzymes that share in their target sites a conserved CCGG tetranucleotide and a cleavage pattern. Structure analysis and biochemical data indicate, that AgeI is a monomer in the apo-form both in the crystal and in solution, however, it binds and cleaves the palindromic target site as a dimer. DNA cleavage mechanism of AgeI is novel among Type IIP restriction endonucleases.
... Of the other members of this novel group, only IRE1 also shows expansion of specificity in addition to activation in the run-on oligomeric form, in its ability to cleave other mRNA as well as its primary target (transcription factor XBP1 mRNA), leading to different biological outcomes (apoptosis vs. stress response) [7]. The proposed biological roles for run-on oligomerization by the different enzymes include rapid activation (or inactivation), storage of inactive enzyme, increase of substrate affinity through larger binding surfaces, and uniquely to SgrAI, sequestration of activated SgrAI on invading phage DNA (which protects the host bacterial genome from secondary site cleavage by activated SgrAI) [8][9][10]. ...
... The x-ray crystal structure of SgrAI has been solved in the low activity, dimeric form bound either to primary or secondary site DNA [11,12]. The structure of the run-on oligomeric form, argued to be the activated form of SgrAI [5], has been described at 8.6 Å using single particle cryo-EM (cryo-EM) reconstruction [10], and shows the association of DNA bound SgrAI dimers in a left-handed helix with contacts between neighboring SgrAI/DNA complexes that include both protein-protein and protein-DNA contacts. The protein-DNA contacts between neighboring complexes occur to the base pairs which flank the recognition site, an observation that explains the dependence of activation of SgrAI on the length or number of these flanking base pairs [5,10,13]. ...
... The structure of the run-on oligomeric form, argued to be the activated form of SgrAI [5], has been described at 8.6 Å using single particle cryo-EM (cryo-EM) reconstruction [10], and shows the association of DNA bound SgrAI dimers in a left-handed helix with contacts between neighboring SgrAI/DNA complexes that include both protein-protein and protein-DNA contacts. The protein-DNA contacts between neighboring complexes occur to the base pairs which flank the recognition site, an observation that explains the dependence of activation of SgrAI on the length or number of these flanking base pairs [5,10,13]. ...
SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single base substitution in either the 1st/8th or the 2nd/7th position of the primary recognition sequence. This modulation of enzyme activity via runon oligomerization is a newly appreciated phenomenon that has been shown for a small but increasing number of enzymes. One outstanding question regarding the mechanistic model for SgrAI is whether or not the activating primary site DNA must be cleaved by SgrAI prior to inducing activation. Herein we show that an uncleavable primary site DNA containing a 3'- S-phosphorothiolate is in fact able to induce activation. In addition, we now show that cleavage of secondary site DNA can be activated to nearly the same degree as primary, provided a sufficient number of flanking base pairs are present. We also show differences in activation and cleavage of the two types of secondary site, and that effects of selected single site substitutions in SgrAI, as well as measured collisional cross-sections from previous work, are consistent with the cryo-electron microscopy model for the run-on activated oligomer of SgrAI bound to DNA.
... SgrAI also assembles into homotetramers, but then goes further and forms 'run-on' oligomers comprising helical filaments of one DNA-bound homodimer after another. Adjacent homodimers are offset ∼90 • , rather than back-to-back, and four homodimers together form almost one turn of a lefthanded spiral, which can comprise up to 18 homodimers and possibly more (367). In this oligomeric form, SgrAI is highly active on both its canonical sequence and on a 'star' sequence, CR|CCGGYN (N = any base). ...
... It implies that the SgrAI homodimer is somewhat asymmetric, such that one subunit consistently recognizes the outer base pair of the recognition sequence, while the other subunit sometimes does not. The homodimer undergoes significant conformational adjustments when it assembles into oligomers (367), and these changes might introduce asymmetry with respect to sequence recognition. ...
... The catalytic complexes of Type IIG enzymes are likely to be large and difficult to solve by crystallography. Alternative approaches such as single particle cryo-electron microscopy and reconstruction (367,377,378), or molecular modeling (379), might prove fruitful in the interim. ...
This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they
are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy,
each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries
of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences.
They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental
levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned,
sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe
the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms
of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising
heterogeneity revealed by comparisons of their sequences and structures.
... Our particular interest in this phenomenon originated with our studies of SgrAI, a type II restriction endonuclease with unusual allosteric behavior, where binding to one type of DNA sequence results in activation of the enzyme to cleave 14 additional DNA sequences 35 . Our investigation into the mechanism responsible for this behavior led to the discovery of filament formation by SgrAI when bound to the activating DNA (which is also a substrate for cleavage of SgrAI known as primary site sequences) 17,[36][37] . The filamentous form recruits additional copies of SgrAI bound to the second type of DNA sequence (secondary sites) 17,38 . ...
... Some other unique features of the SgrAI system are also found, including filament formation being stimulated by both substrate and product (of the primary site sequence), participation of the substrate (and product) in stabilization of the filament via direct interactions with neighboring protomers, and the requirement to dissociate from the filament in order to release product 17,[37][38][50][51][52] . We found that the dynamics of SgrAI enzymes into and out of the filament is sufficiently fast to prevent trapping of the product, despite this requirement. ...
Filament formation by non-cytoskeletal enzymes has been known for decades, yet only relatively recently has its wide-spread role in enzyme regulation and biology come to be appreciated. This comprehensive review summarizes what is known for each enzyme confirmed to form filamentous structures in vitro, and for the many that are known only to form large self-assemblies within cells. For some enzymes, studies describing both the in vitro filamentous structures and cellular self-assembly formation are also known and described. Special attention is paid to the detailed structures of each type of enzyme filament, as well as the roles the structures play in enzyme regulation and in biology. Where it is known or hypothesized, the advantages conferred by enzyme filamentation are reviewed. Finally, the similarities, differences, and comparison to the SgrAI system are also highlighted.
... However, assemblies containing many more DBDs were shown by analytical ultracentrifugation and ion-mobility mass spectrometry (Ma et al., 2013;Park et al., 2010). Negative-stain EM revealed filaments of varied lengths with left-handed helical symmetry that we call run-on oligomers (ROO) (Lyumkis et al., 2013). ...
... The ROO filament structure was previously resolved to approximately nanometer resolution by cryo-EM and helical reconstruction, and revealed left-handed helical symmetry with approximately 4 DBDs per turn (Lyumkis et al., 2013). This structure inspired a low-resolution mechanistic model for the enzymatic behavior of SgrAI, wherein binding to primary-site DNA induces a conformational change that favors ROO filament formation, which in turn stabilizes the activated enzyme state capable of accelerated DNA cleavage ( Figure 1A). ...
Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the cryoelectron microscopy (cryo-EM) structure of the filament of SgrAI in its activated configuration. The structural data illuminate the mechanistic origin of hyperaccelerated DNA cleavage activity and suggests how indirect DNA sequence readout within filamentous SgrAI may enable recognition of substantially more nucleotide sequences than its low-activity form, thereby altering and partially relaxing its DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI's catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.
... The wide diversity of restriction enzymes 18 , from the smallest dimeric PvuII 19 , to tetrameric Type IIF enzymes 20 , and the polymerized SgrAI 21 , make them versatile tools for laboratory experimentation, and fascinating subjects for studies of molecular architecture 22 . Here we show structurally that the modification-dependent restriction enzyme AspBHI comprises two domains, one typically eukaryotic and the other typically prokaryotic. ...
... For plasmid digestions, 100 to 300 ng of DNA was digested with 1-5 mg of AspBHI (1 mg ml 21 ) in NEB buffer 4 in the presence of 15 mM of a self-annealed stem-loop activator (59 CTCCMAGGATCTTTTTTGATCMTGGGAG-39 where M 5 5mC) 4 . Adding an activator with the recognition sequence in trans can accelerate the slow reactions by the AspBHI family members 4 . ...
The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.
... The copyright holder for this preprint (which this version posted May 1, 2024. ; https://doi.org/10.1101/2024.05.01.592068 doi: bioRxiv preprint structures of SgrAI in both filamentous and non-filamentous states to understand the structural origins of the observed filamentation-induced activation and modulation of specificity (23)(24)(25)(26)34). The two new structures presented here clarify key outstanding questions regarding the activated DNA cleavage mechanism mediated by SgrAI. ...
Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments. SgrAI filamentation increases enzymatic activity by up to three orders of magnitude and additionally expands its DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-electron microscopy (cryo-EM). The first structure, resolved to 3.3 Angstrom, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Angstrom, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes.
... To better understand their mechanisms of cleavage and sequence recognition, the structures of almost 40 Type II REases bound to DNA have been solved by X-ray crystallography. Most act as dimers (EcoRI (4), EcoRV (5), BamHI (6), PvuII (7) and BglI (8)) or tetramers (Cfr10I (9), NgoMIV (10), SfiI (11), SgrAI (12), MspJI (13,14)) of identical subunits. Each subunit contains one catalytic site such that when the dimer binds to its target sequence, one catalytic site cleaves one DNA strand and the other site cleaves the other strand. ...
HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5'-GCG↓C-3' in duplex DNA and cleaves ('↓') to produce fragments with 2-base, 3'-overhangs. We determined the structure of HhaI in complex with cognate DNA at an ultra-high atomic resolution of 1.0 Å. Most restriction enzymes act as dimers with two catalytic sites, and cleave the two strands of duplex DNA simultaneously, in a single binding event. HhaI, in contrast, acts as a monomer with only one catalytic site, and cleaves the DNA strands sequentially, one after the other. HhaI comprises three domains, each consisting of a mixed five-stranded β sheet with a defined function. The first domain contains the catalytic-site; the second contains residues for sequence recognition; and the third contributes to non-specific DNA binding. The active-site belongs to the 'PD-D/EXK' superfamily of nucleases and contains the motif SD-X11-EAK. The first two domains are similar in structure to two other monomeric restriction enzymes, HinP1I (G↓CGC) and MspI (C↓CGG), which produce fragments with 5'-overhangs. The third domain, present only in HhaI, shifts the positions of the recognition residues relative to the catalytic site enabling this enzyme to cleave the recognition sequence at a different position. The structure of M.HhaI, the biological methyltransferase partner of HhaI, was determined earlier. Together, these two structures represent the first natural pair of restriction-modification enzymes to be characterized in atomic detail.
... The simplest correspond to monomers or homodimers that recognize palindromic or nearpalindromic DNA targets, and cut at equivalent positions within each DNA strand (22)(23)(24)(25). More complex Type II enzymes act as homo-tetramers (26)(27)(28)(29)(30)(31) or bind to target sites in one structural form, and then oligomerize 'transiently' in order to cleave the DNA (32)(33)(34)(35). In both cases, binding to two or more target sites simultaneously is often a prerequisite for efficient cleavage (36). ...
BbvCI, a Type IIT restriction endonuclease, recognizes and cleaves the seven base pair sequence 5'-CCTCAGC-3', generating 3-base, 5'-overhangs. BbvCI is composed of two protein subunits, each containing one catalytic site. Either site can be inactivated by mutation resulting in enzyme variants that nick DNA in a strand-specific manner. Here we demonstrate that the holoenzyme is labile, with the R1 subunit dissociating at low pH. Crystallization of the R2 subunit under such conditions revealed an elongated dimer with the two catalytic sites located on opposite sides. Subsequent crystallization at physiological pH revealed a tetramer comprising two copies of each subunit, with a pair of deep clefts each containing two catalytic sites appropriately positioned and oriented for DNA cleavage. This domain organization was further validated with single-chain protein constructs in which the two enzyme subunits were tethered via peptide linkers of variable length. We were unable to crystallize a DNA-bound complex; however, structural similarity to previously crystallized restriction endonucleases facilitated creation of an energy-minimized model bound to DNA, and identification of candidate residues responsible for target recognition. Mutation of residues predicted to recognize the central C:G base pair resulted in an altered enzyme that recognizes and cleaves CCTNAGC (N = any base).
... At high concentrations BsaWI oligomers are able to bind DNA but do not cleave it. also a dimer in the apo-form, however after binding to a specific target site it forms so called 'run-on' oligomers, which show relaxed sequence specificity (8,15,48,49). The suggested mechanism for BsaWI shares common features both with dimeric Ecl18kI/SgrAI and tetrameric Type IIF enzymes NgoMIV/Bse634I/Cfr10I and SfiI. ...
Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5'-W/CCGGW-3' (W stands for A or T, '/' denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an 'open' configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes.
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
... Lastly, the LC and RGG-ZnF-RGG domains interact with each other in trans in an RNA-dependent manner (53). Analogous allosteric regulation of nucleic acid binding proteins that triggers self-assembly has been proposed for the bacterial SgrAI and eukaryotic IreI proteins (75,76). ...
Members of the FET protein family, consisting of FUS, EWSR1, and TAF15, bind to RNA and contribute to the control of transcription, RNA processing, and the cytoplasmic fates of messenger RNAs in metazoa. FET proteins can also bind DNA, which may be important in transcription and DNA damage responses. FET proteins are of medical interest because chromosomal rearrangements of their genes promote various sarcomas and because point mutations in FUS or TAF15 can cause neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. Recent results suggest that both the normal and pathological effects of FET proteins are modulated by low-complexity or prion-like domains, which can form higher-order assemblies with novel interaction properties. Herein, we review FET proteins with an emphasis on how the biochemical properties of FET proteins may relate to their biological functions and to pathogenesis. Expected final online publication date for the Annual Review of Biochemistry Volume 84 is June 02, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... They occur naturally in bacteria and archaea, and come in numerous different forms (64), from simple monomers [e.g. MspI (65)] and dimers [BamHI (66,67); PvuII (68)], to tetramers [NgoMIV (69); SfiI (70)], polymers [SgrAI (71)], and complex enzymes with allosteric regulatory domains [NaeI (72,73); EcoRII (74)]. The proteins can comprise one domain [HindIII (75)], two domains [FokI (61,76)], three [MmeI (77)] or more [TstI (78)]. ...
AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ∼70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ∼22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.
This review documents investigations leading to the unprecedented discovery of filamentation as a mode of enzyme regulation in the type II restriction endonuclease SgrAI. Filamentation is defined here as linear or helical polymerization of a single enzyme as occurs for SgrAI, and has now been shown to occur in many other enzyme systems, including conserved metabolic enzymes. In the case of SgrAI, filamentation activates the DNA cleavage rate by up to 1000-fold and also alters the enzyme's DNA sequence specificity. The investigations began with the observation that SgrAI cleaves two types of recognition sequences, primary and secondary, but cleaves the secondary sequences only when present on the same DNA as at least one primary. DNA cleavage rate measurements showed how the primary sequence is both a substrate and an allosteric effector of SgrAI. Biophysical measurements indicated that the activated form of SgrAI, stimulated by binding to the primary sequence, consisted of varied numbers of the SgrAI bound to DNA. Structural studies revealed the activated state of SgrAI as a left-handed helical filament which stabilizes an altered enzyme conformation, which binds a second divalent cation in the active site. Efforts to determine the mechanism of DNA sequence specificity alteration are ongoing and current models are discussed. Finally, global kinetic modeling of the filament mediated DNA cleavage reaction and simulations of in vivo activity suggest that the filament mechanism evolved to rapidly cleave invading DNA while protecting the Streptomyces host genome.
Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg²⁺ and define how the Mg²⁺ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg²⁺ in the SgrAI active site as a direct result of filamentation induced conformational changes.
Background:
Restriction endonucleases belong to prokaryotic restriction-modification systems, that protect host cells from invading DNA. Type II restriction endonucleases recognize short 4-8 bp sequences in the target DNA and cut both DNA strands producing double strand breaks. Type II restriction endonuclease Kpn2I cleaves 5'-T/CCGGA DNA sequence ("/" marks the cleavage position). Analysis of protein sequences suggested that Kpn2I belongs to the CCGG-family, which contains ten enzymes that recognize diverse nucleotides outside the conserved 5'-CCGG core and share similar motifs for the 5'-CCGG recognition and cleavage.
Methods:
We solved a crystal structure of Kpn2I in a DNA-free form at 2.88 Å resolution. From the crystal structure we predicted active center and DNA recognition residues and tested them by mutational analysis. We estimated oligomeric state of Kpn2I by SEC-MALS and performed plasmid DNA cleavage assay to elucidate DNA cleavage mechanism.
Results:
Structure comparison confirmed that Kpn2I shares a conserved active site and structural determinants for the 5'-CCGG tetranucleotide recognition with other restriction endonucleases of the CCGG-family. Guided by structural similarity between Kpn2I and the CCGG-family restriction endonucleases PfoI and AgeI, Kpn2I residues involved in the outer base pair recognition were proposed.
Conclusions:
Kpn2I is an orthodox Type IIP restriction endonuclease, which acts as a dimer. Kpn2I shares structural similarity to the CCGG-family restriction endonucleases PfoI, AgeI and PspGI.
General significance:
The Kpn2I structure concluded the studies of the CCGG-family, covering detailed structural and biochemical characterization of eleven restriction enzymes and their complexes with DNA.
The structural and dynamical characterization of biomolecules holds central importance in the endeavor to understand the molecular mechanisms regulating living systems. However, owing to the inherent heterogeneity of biomolecular interactions within cells, it is often difficult to understand the overall structure and dynamics of biomolecules using any experimental method in isolation. In this regard, hybrid methods that combine data from multiple experiments in order to generate a comprehensive model of biomolecular complexes, have gained prominence in the last few years. In this review, we discuss the advancements in hybrid methods, with a particular focus on the role of computation in their development and application. We further outline the future directions that hybrid methods are likely to take, regarding the advancements in techniques like X-ray Free Electron Laser single particle imaging and Electron Cryo-Tomography. Finally, we conclude the review by highlighting the future goals of broader consensus and collaboration within the integrative/hybrid structural biology community, and for disseminating the data generated by hybrid modeling efforts.
Hybrid approaches combine computational modeling techniques with low-resolution structural data. Such approaches have proven to be powerful tools to obtain new 3D structural and dynamical information on biological systems. Currently, major applications focus on cryo-EM data. Methods and some applications to construct atomic structural models of new functional states will be reviewed. In addition, possible extension to data from X-ray free electron laser, which currently provides low-resolution data, will be discussed.
In this issue of Structure, Lyumkis and colleagues describe a high resolution structure of a polymerized form of the SgrAI restriction enzyme, which shows that it forms a helical assembly with four enzyme molecules per turn of the helix. The DNA is arranged on the periphery of the protein helix pointing away from the helical axis.
REBASE is a comprehensive database of information about restriction enzymes, DNA methyltransferases and related proteins involved
in the biological process of restriction–modification (R–M). It contains fully referenced information about recognition and
cleavage sites, isoschizomers, neoschizomers, commercial availability, methylation sensitivity, crystal and sequence data.
Experimentally characterized homing endonucleases are also included. The fastest growing segment of REBASE contains the putative
R–M systems found in the sequence databases. Comprehensive descriptions of the R–M content of all fully sequenced genomes
are available including summary schematics. The contents of REBASE may be browsed from the web (http://rebase.neb.com) and selected compilations can be downloaded by ftp (ftp.neb.com). Additionally, monthly updates can be requested via email.
Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, an essential substrate for fatty acid biosynthesis and
a potent inhibitor of fatty acid oxidation. Here, we provide evidence that glutamate may be a physiologically relevant activator
of ACC. Glutamate induced the activation of both major isoforms of ACC, prepared from rat liver, heart, or white adipose tissue.
In agreement with previous studies, a type 2A protein phosphatase contributed to the effects of glutamate on ACC. However,
the protein phosphatase inhibitor microcystin LR did not abolish the effects of glutamate on ACC activity. Moreover, glutamate
directly activated purified preparations of ACC when protein phosphatase activity was excluded. Phosphatase-independent ACC
activation by glutamate was also reflected by polymerization of the enzyme as judged by size-exclusion chromatography. The
sensitivity of ACC to direct activation by glutamate was diminished by treatment in vitro with AMP-activated protein kinase or cAMP-dependent protein kinase or by β-adrenergic stimulation of intact adipose tissue.
We conclude that glutamate, an abundant intracellular amino acid, induces ACC activation through complementary actions as
a phosphatase activator and as a direct allosteric ligand for dephosphorylated ACC. This study supports the general hypothesis
that amino acids fulfill important roles as signal molecules as well as intermediates in carbon and nitrogen metabolism.
SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits self-modulation of DNA cleavage activity and sequence specificity. Previous studies have shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of SgrAI:DNA as well as the basic building block of the oligomers, has not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI/DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD) with one SgrAI dimer bound to two pre-cleaved duplex DNA molecules each containing one half of the SgrAI primary recognition sequence. The oligomers contain variable numbers of DBDs with as many as 19 DBDs. Observation of the large oligomers shows that nano-electrospray ionization (nano-ESI) can preserve the proposed activated form of an enzyme. Finally, the collision cross section (CCS) of the SgrAI/DNA oligomers measured by IM-MS was found to have a linear relationship with the number of DBD in each oligomer suggesting a regular, repeating structure.
Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its evolutionarily conserved large size, Ltn1 is characterized by the presence of a conserved N terminus, HEAT/ARM repeats predicted to comprise the majority of the protein, and a C-terminal catalytic RING domain, although the protein's exact structure is unknown. We used numerous single-particle EM strategies to characterize Ltn1's structure based on negative stain and vitreous ice data. Two-dimensional classifications and subsequent 3D reconstructions of electron density maps show that Ltn1 has an elongated form and presents a continuum of conformational states about two flexible hinge regions, whereas its overall architecture is reminiscent of multisubunit cullin-RING ubiquitin ligase complexes. We propose a model of Ltn1 function based on its conformational variability and flexibility that describes how these features may play a role in cotranslational protein quality control.
Identification of homogeneous subsets of images in a macromolecular electron microscopy (EM) image data set is a critical step in single-particle analysis. The task is handled by iterative algorithms, whose performance is compromised by the compounded limitations of image alignment and K-means clustering. Here we describe an approach, iterative stable alignment and clustering (ISAC) that, relying on a new clustering method and on the concepts of stability and reproducibility, can extract validated, homogeneous subsets of images. ISAC requires only a small number of simple parameters and, with minimal human intervention, can eliminate bias from two-dimensional image clustering and maximize the quality of group averages that can be used for ab initio three-dimensional structural determination and analysis of macromolecular conformational variability. Repeated testing of the stability and reproducibility of a solution within ISAC eliminates heterogeneous or incorrect classes and introduces critical validation to the process of EM image clustering.
The study of yeast cell morphology requires consistent identification of cell cycle phases based on cell bud size. A computer-based image processing algorithm is designed to automatically classify microscopic images of yeast cells in a microfluidic channel environment. The images were enhanced to reduce background noise, and a robust segmentation algorithm is developed to extract geometrical features including compactness, axis ratio, and bud size. The features are then used for classification, and the accuracy of various machine-learning classifiers is compared. The linear support vector machine, distance-based classification, and k-nearest-neighbor algorithm were the classifiers used in this experiment. The performance of the system under various illumination and focusing conditions were also tested. The results suggest it is possible to automatically classify yeast cells based on their morphological characteristics with noisy and low-contrast images.
SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-activation with expansion of DNA-sequence specificity. The three-dimensional crystal structures of SgrAI bound to cleaved primary-site DNA and Mg²⁺ and bound to secondary-site DNA with either Mg²⁺ or Ca²⁺ are presented. All three structures show a conformation of enzyme and DNA similar to the previously determined dimeric structure of SgrAI bound to uncleaved primary-site DNA and Ca²⁺ [Dunten et al. (2008), Nucleic Acids Res. 36, 5405–5416], with the exception of the cleaved bond and a slight shifting of the DNA in the SgrAI/cleaved primary-site DNA/Mg²⁺ structure. In addition, a new metal ion binding site is located in one of the two active sites in this structure, which is consistent with proposals for the existence of a metal-ion site near the 3′-O leaving group.
SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of cleavage activity and sequence specificity. Previous studies have shown that DNA bound dimers of SgrAI oligomerize into an activated form with higher DNA cleavage rates, although previously determined crystal structures of SgrAI bound to DNA show only the DNA bound dimer. A new crystal structure of the type II restriction endonuclease SgrAI bound to DNA and Ca(2+) is now presented, which shows the close association of two DNA bound SgrAI dimers. This tetrameric form is unlike those of the homologous enzymes Cfr10I and NgoMIV and is formed by the swapping of the amino-terminal 24 amino acid residues. Two mutations predicted to destabilize the swapped form of SgrAI, P27W and P27G, have been made and shown to eliminate both the oligomerization of the DNA bound SgrAI dimers as well as the allosteric stimulation of DNA cleavage by SgrAI. A mechanism involving domain swapping is proposed to explain the unusual allosteric properties of SgrAI via association of the domain swapped tetramer of SgrAI bound to DNA into higher order oligomers.
The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate-mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity. CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four novel filaments greatly expands the number of known intracellular filament networks and has broad implications for our understanding of how cells organize biochemical activities in the cytoplasm.
Filament-forming cytoskeletal proteins are essential for the structure and organization of all cells. Bacterial homologues of the major eukaryotic cytoskeletal families have now been discovered, but studies suggest that yet more remain to be identified. We demonstrate that the metabolic enzyme CTP synthase (CtpS) forms filaments in Caulobacter crescentus. CtpS is bifunctional, as the filaments it forms regulate the curvature of C. crescentus cells independently of its catalytic function. The morphogenic role of CtpS requires its functional interaction with the intermediate filament, crescentin (CreS). Interestingly, the Escherichia coli CtpS homologue also forms filaments both in vivo and in vitro, suggesting that CtpS polymerization may be widely conserved. E. coli CtpS can replace the enzymatic and morphogenic functions of C. crescentus CtpS, indicating that C. crescentus has adapted a conserved filament-forming protein for a secondary role. These results implicate CtpS as a novel bifunctional member of the bacterial cytoskeleton and suggest that localization and polymerization may be important properties of metabolic enzymes.
Aberrant folding of proteins in the endoplasmic reticulum activates the bifunctional transmembrane kinase/endoribonuclease Ire1. Ire1 excises an intron from HAC1 messenger RNA in yeasts and Xbp1 messenger RNA in metozoans encoding homologous transcription factors. This non-conventional mRNA splicing event initiates the unfolded protein response, a transcriptional program that relieves the endoplasmic reticulum stress. Here we show that oligomerization is central to Ire1 function and is an intrinsic attribute of its cytosolic domains. We obtained the 3.2-A crystal structure of the oligomer of the Ire1 cytosolic domains in complex with a kinase inhibitor that acts as a potent activator of the Ire1 RNase. The structure reveals a rod-shaped assembly that has no known precedence among kinases. This assembly positions the kinase domain for trans-autophosphorylation, orders the RNase domain, and creates an interaction surface for binding of the mRNA substrate. Activation of Ire1 through oligomerization expands the mechanistic repertoire of kinase-based signalling receptors.
The three-dimensional X-ray crystal structure of the ‘rare cutting’ type II restriction endonuclease SgrAI bound to cognate
DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca2+ bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from
the DNA. These sites are differentially occupied by Mn2+, with strong binding at the protein–DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved
in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar
to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central
CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar
to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence
may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact
to the G from an arginine side chain located in a region unique to SgrAI.
We have isolated SgrAI, a novel class-Il restriction endonuclease
from Streptomyces griseus (Soil Microbiology Associates, Inc.)
recognizing the new octanucleotide palindromic sequence
5'-CR/CCGGYG-3' generating 5'-protruding CCGGtetranucleotides
(1). SgrAI complements Notl and Sfi1 (2) both
recognizing octanucleotide sequences; the novel enzyme may be
a useful tool for rare cutting approaches (3).
A comparison of cleavage patterns experimentally obtained
with SgrAI on standard lambda, Ad-2, SV40, OX174, Ml3mp7,
pBR322 and pBR328 DNAs of known nucleotide sequence (Fig.
1, lanes 2-8) with computer-derived mapping data (4) predicts
the sequence 5'-CRCCGGYG-3'. Digestion of lambda DNA
yielded in 7 fragments of approximately 17000, 15000, 7100,
4200, 2800, 1600 and 1300 bp, which correlate with the
computer-derived lengths of 16678, 14850, 7064, 4190, 2775,
1616 and 1321 bp. Ad-2 yields in 6 fragments; pBR322 and
pBR328 are linearized. The other DNAs are not cut by SgrAI.
The cut positions within the SgrAI recognition site were
determined in two independent experiments according to the
enzymatic sequencing approach described in (5). An
M13mpl8-derivative with an insert containing an SgrAI cleavage
site was used for enzymatic sequencing reactions starting with
a 5'-phosphorylated M13 universal sequencing primer. In a
parallel reaction, the same primer, [32P]-endlabeled with T4
PNK and [Fy-32P]ATP, was annealed to the template, and the
labeled primer was extended by treatment with Klenow enzyme
and all four dNTPs through the SgrAI site. The double-stranded
DNA was used as substrate for SgrAI to produce 5'-endlabeled
DNA fragments comparable to the sequencing ladder. Samples
were analyzed without or with (-/+) further incubation with
T4 DNAP and all four dNTPs by electrophoresis and subsequent
autoradiography (Fig. 2). In a first experiment with the SgrAI
T4 DNAP treatment, the observed band shift refers to G(6) of
the recognition sequence 5'-CA/CCGGTG-3'. In a second
experiment the SgrAI-specific band comigrated with A(2); the
observed band shift refers to G(6) of the recognition sequence
5'-CA/CCGGCG-3'.
From the mapping and sequencing data the specificity of SgrAI
is concluded as:
5'-CR/CCGG-YG-3'
3'-GY-GGCC/RC-5'.
Type II restriction endonucleases usually recognize 4–6-base pair (bp) sites on DNA and cleave each site in a separate reaction.
A few type II endonucleases have 8-bp recognition sites, but these seem unsuited for restriction, since their sites are rare
on most DNA. Moreover, only one endonuclease that recognizes a target containing 8 bp has been examined to date, and this
enzyme,SfiI, needs two copies of this site for its DNA cleavage reaction. In this study, several endonucleases with 8-bp sites were
tested on plasmids that have either one or two copies of the relevant sequence to determine if they also need two sites. SgfI,SrfI, FseI, PacI, PmeI,Sse8781I, and SdaI all acted through equal and independent reactions at each site. AscI cleaved the DNA with one site at the same rate as that with two sites but acted processively on the latter. In contrast,
SgrAI showed a marked preference for the plasmid with two sites and cleaved both sites on this DNA in a concerted manner, like
SfiI. Endonucleases that require two copies of an 8-bp sequence may be widespread in nature, where, despite this seemingly inappropriate
requirement, they may function in DNA restriction.
More than 3000 type II restriction endonucleases have been discovered. They recognize short, usually palindromic, sequences of 4-8 bp and, in the presence of Mg(2+), cleave the DNA within or in close proximity to the recognition sequence. The orthodox type II enzymes are homodimers which recognize palindromic sites. Depending on particular features subtypes are classified. All structures of restriction enzymes show a common structural core comprising four beta-strands and one alpha-helix. Furthermore, two families of enzymes can be distinguished which are structurally very similar (EcoRI-like enzymes and EcoRV-like enzymes). Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone. In contrast, specific binding is characterized by an intimate interplay between direct (interaction with the bases) and indirect (interaction with the backbone) readout. Typically approximately 15-20 hydrogen bonds are formed between a dimeric restriction enzyme and the bases of the recognition sequence, in addition to numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may also be water mediated. The recognition process triggers large conformational changes of the enzyme and the DNA, which lead to the activation of the catalytic centers. In many restriction enzymes the catalytic centers, one in each subunit, are represented by the PD. D/EXK motif, in which the two carboxylates are responsible for Mg(2+) binding, the essential cofactor for the great majority of enzymes. The precise mechanism of cleavage has not yet been established for any enzyme, the main uncertainty concerns the number of Mg(2+) ions directly involved in cleavage. Cleavage in the two strands usually occurs in a concerted fashion and leads to inversion of configuration at the phosphorus. The products of the reaction are DNA fragments with a 3'-OH and a 5'-phosphate.
The primary target of SgrAI restriction endonuclease is a multiple sequence of the form 5'-CPu/CCGGPyG. Previous work had indicated that SgrAI must bind two recognition sites simultaneously for catalysis [Bilcock, D. T., Daniels, L. E., Bath, A. J. & Halford, S. E. (1999) J. Biol. Chem. 274, 36379-36386]. In the present study, SgrAI is shown to cleave not only its canonical sequences, but also the sequences 5'-CPuCCGGPy(A,T,C) and 5'-CPuCCGGGG, both referred to as secondary sequences. On plasmid pSK7, SgrAI cleaves secondary sites 26-fold slower than the canonical site. However, the same plasmid, but without the canonical site, is cleaved 200-fold slower. We show that DNA termini generated by cleaving the canonical site for SgrAI assist in the cleavage of secondary sites. The SgrAI-termini in cis with respect to secondary site are markedly preferred over those in trans. The SgrAI-termini provided in a form of oligonucleotide duplex are also shown to stimulate canonical site cleavage. At a 40-fold molar excess of the SgrAI-termini over substrate, the SgrAI specificity is shown to improve by two orders of magnitude, because of concurrent 10-fold increase in the cleavage of canonical site and 50-fold decrease in the cleavage of secondary sites. The unconventional reaction pathway by which SgrAI utilizes the self-generated DNA termini to cleave its DNA targets has not been observed hitherto among type II restriction endonucleases. Based on our work and previous reports, a pathway of DNA binding and cleavage by the SgrAI restriction endonuclease is proposed.
SgrAI restriction endonuclease cooperatively interacts and cleaves two target sites that include both the canonical sites, CPuCCGGPyG,
and the secondary sites, CPuCCGGPy(A/T/C). It has been observed that the cleaved canonical sites stimulate SgrAI cleavage at the secondary sites. Equilibrium binding studies show that SgrAI binds to its canonical sites with a high affinity (Ka = 4-8 × 1010 m-1) and that it has a 15-fold lower affinity for the cleaved canonical sites and a 30-fold lower affinity for the secondary
sites. Steady-state kinetics reveals substrate cooperativity for SgrAI cleavage on both canonical and secondary sites. The specificity of SgrAI for the secondary site CACCGGCT, as measured by kcat/K is about 500-fold lower than that for the canonical site CACCGGCG, but this difference is reduced to 10-fold in the presence
of the cleaved canonical sites. The efficiency of canonical site cleavage also increases by 3-fold when the cleaved canonical
sites are present in the reaction. Furthermore, the substrate cooperativity for SgrAI cleavage is abolished for both types of sites in the presence of cleaved canonical sites. These results indicate that target
site cleavage occurs via a coordinated interaction of two SgrAI protein subunits, where the subunit bound to the cleaved site stimulates the cleavage of the uncut site bound by the other
subunit. The free subunits of SgrAI have the flexibility to bind different target sites and, consequently, assemble into various catalytically active complexes,
which differ in their catalytic efficiencies.
REBASE is a comprehensive and fully curated database of information about the components of restriction-modification (RM) systems. It contains fully referenced information about recognition and cleavage sites for both restriction enzymes and methyltransferases as well as commercial availability, methylation sensitivity, crystal and sequence data. All genomes that are completely sequenced are analyzed for RM system components, and with the advent of PacBio sequencing, the recognition sequences of DNA methyltransferases (MTases) are appearing rapidly. Thus, Type I and Type III systems can now be characterized in terms of recognition specificity merely by DNA sequencing. The contents of REBASE may be browsed from the web http://rebase.neb.com and selected compilations can be downloaded by FTP (ftp.neb.com). Monthly updates are also available via email.
Evaluation of the electrostatic properties of biomolecules has become a standard practice in molecular biophysics. Foremost
among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods
for solving this equation have limited the scope of accurate electrostatic calculations to relatively small biomolecular systems.
Here we present the application of numerical methods to enable the trivially parallel solution of the Poisson-Boltzmann equation
for supramolecular structures that are orders of magnitude larger in size. As a demonstration of this methodology, electrostatic
potentials have been calculated for large microtubule and ribosome structures. The results point to the likely role of electrostatics
in a variety of activities of these structures.
Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence.
In contrast to oligodeoxynucleotides, protein:DNA complexes crystallize from a broad range of precipitants and conditions, much as proteins by themselves. There are, however, a number of factors that should be considered, at least in the early stages of cocrystallization attempts. These include the length and construction of the oligodeoxynucleotide itself, a pH near or below neutrality, a stoichiometric excess of DNA, and di- and polyvalent cations. By far the more important of these factors are the length of the DNA fragment and the nature of the terminal nucleotides. Unfortunately, experience suggests that, in general, the length and construction of the DNA fragment for optimal crystal growth cannot be predicted in advance. A systematic search of cocrystallization conditions with different DNA fragments will normally be required. It is important to avoid sequences that provide possible subsites within the fragment or at junctions between fragments related by translations. Sample purity, especially that of the DNA, can also have important effects. Detailed protocols for the purification of chemically synthesized fragments are suggested. Finally, a set of conditions for initial cocrystallization trials with a new DNA binding protein is suggested.
Abramoff, M.D., Magelhaes, P.J., Ram, S.J. "Image Processing with ImageJ". Biophotonics International, volume 11, issue 7, pp. 36-42, 2004.
Restriction endonucleases are enzymes which recognize short DNA sequences and cleave the DNA in both strands. Depending on the enzymological properties different types are distinguished. Type II restriction endonucleases are homodimers which recognize short palindromic sequences 4–8 bp in length and, in the presence of Mg2+, cleave the DNA within or next to the recognition site. They are capable of non-specific binding to DNA and make use of linear diffusion to locate their target site. Binding and recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10–12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic centers. The precise mechanism of cleavage has not yet been established for any restriction endonuclease. Currently two models are discussed: the substrate-assisted catalysis mechanism and the two-metal-ion mechanism. Structural similarities identified between EcoRI, EcoRV, BamHI, PvuII and Cfr10I suggest that many type II restriciton endonucleases are not only functionally but also evolutionarily related.
We present a major revision of the iterative helical real-space refinement (IHRSR) procedure and its implementation in the SPARX single particle image processing environment. We built on over a decade of experience with IHRSR helical structure determination and we took advantage of the flexible SPARX infrastructure to arrive at an implementation that offers ease of use, flexibility in designing helical structure determination strategy, and high computational efficiency. We introduced the 3D projection matching code which now is able to work with non-cubic volumes, the geometry better suited for long helical filaments, we enhanced procedures for establishing helical symmetry parameters, and we parallelized the code using distributed memory paradigm. Additional features include a graphical user interface that facilitates entering and editing of parameters controlling the structure determination strategy of the program. In addition, we present a novel approach to detect and evaluate structural heterogeneity due to conformer mixtures that takes advantage of helical structure redundancy.
Bacteria, the most abundant organisms on the planet, are outnumbered by a factor of 10 to 1 by phages that infect them. Faced with the rapid evolution and turnover of phage particles, bacteria have evolved various mechanisms to evade phage infection and killing, leading to an evolutionary arms race. The extensive co-evolution of both phage and host has resulted in considerable diversity on the part of both bacterial and phage defensive and offensive strategies. Here, we discuss the unique and common features of phage resistance mechanisms and their role in global biodiversity. The commonalities between defense mechanisms suggest avenues for the discovery of novel forms of these mechanisms based on their evolutionary traits.
SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of DNA activity and sequence specificity. Precleaved primary site DNA has been shown to be an allosteric effector [Hingorani-Varma, K., and Bitinaite, J. (2003) J. Biol. Chem. 278, 40392-40399], stimulating cleavage of both primary (CR|CCGGYG, where the vertical bar indicates a cut site, R denotes A or G, and Y denotes C or T) and secondary [CR|CCGGY(A/C/T) and CR|CCGGGG] site DNA sequences. The fact that DNA is the allosteric effector of this endonuclease suggests at least two DNA binding sites on the functional SgrAI molecule, yet crystal structures of SgrAI [Dunten, P. W., et al. (2008) Nucleic Acids Res. 36, 5405-5416] show only one DNA duplex bound to one dimer of SgrAI. We show that SgrAI forms species larger than dimers or tetramers [high-molecular weight species (HMWS)] in the presence of sufficient concentrations of SgrAI and its primary site DNA sequence that are dependent on the concentration of the DNA-bound SgrAI dimer. Analytical ultracentrifugation indicates that the HMWS is heterogeneous, has sedimentation coefficients of 15-20 s, and is composed of possibly 4-12 DNA-bound SgrAI dimers. SgrAI bound to secondary site DNA will not form HMWS itself but can bind to HMWS formed with primary site DNA and SgrAI. Uncleaved, as well as precleaved, primary site DNA is capable of stimulating HMWS formation. Stimulation of DNA cleavage by SgrAI, at primary as well as secondary sites, is also dependent on the concentration of primary site DNA (cleaved or uncleaved) bound SgrAI dimers. SgrAI bound to secondary site DNA does not have significant stimulatory activity. We propose that the oligomers of DNA-bound SgrAI (i.e., HMWS) are the activated, or activatable, forms of the enzyme.
It has been more than 50 years since the elucidation of the structure of double-helical DNA. Despite active research and progress in DNA biology and biochemistry, much remains to be learned in the field of DNA biophysics. Predicting the sequence-dependent curvature and flexibility of DNA is difficult. Applicability of the conventional worm-like chain polymer model of DNA has been challenged. The fundamental forces responsible for the remarkable resistance of DNA to bending and twisting remain controversial. The apparent 'softening' of DNA measured in vivo in the presence of kinking proteins and superhelical strain is incompletely understood. New methods and insights are being applied to these problems. This review places current work on DNA biophysics in historical context and illustrates the ongoing interplay between theory and experiment in this exciting field.
Cryo-electron microscopy produces 3D density maps of molecular machines, which consist of various molecular components such as proteins and RNA. Segmentation of individual components in such maps is a challenging task, and is mostly accomplished interactively. We present an approach based on the immersive watershed method and grouping of the resulting regions using progressively smoothed maps. The method requires only three parameters: the segmentation threshold, a smoothing step size, and the number of smoothing steps. We first apply the method to maps generated from molecular structures and use a quantitative metric to measure the segmentation accuracy. The method does not attain perfect accuracy, however it produces single or small groups of regions that roughly match individual proteins or subunits. We also present two methods for fitting of structures into density maps, based on aligning the structures with single regions or small groups of regions. The first method aligns centers and principal axes, whereas the second aligns centers and then rotates the structure to find the best fit. We describe both interactive and automated ways of using these two methods. Finally, we show segmentation and fitting results for several experimentally-obtained density maps.
Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a "toolbox" of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.
Actin filaments and microtubules polymerize and depolymerize by adding and removing subunits at polymer ends, and these dynamics
drive cytoplasmic organization, cell division, and cell motility. Since Wegner proposed the treadmilling theory for actin
in 1976, it has largely been assumed that the chemical state of the bound nucleotide determines the rates of subunit addition
and removal. This chemical kinetics view is difficult to reconcile with observations revealing multiple structural states
of the polymer that influence polymerization dynamics but that are not strictly coupled to the bound nucleotide state. We
refer to these phenomena as “structural plasticity” and discuss emerging evidence that they play a central role in polymer
dynamics and function.
Despite the importance of subcellular localization for cellular activities, the lack of high-throughput, high-resolution imaging and quantitation methodologies has limited genomic localization analysis to a small number of archival studies focused on C-terminal fluorescent protein fusions. Here, we develop a high-throughput pipeline for generating, imaging, and quantitating fluorescent protein fusions that we use for the quantitative genomic assessment of the distributions of both N- and C-terminal fluorescent protein fusions. We identify nearly 300 localized Caulobacter crescentus proteins, up to 10-fold more than were previously characterized. The localized proteins tend to be involved in spatially or temporally dynamic processes and proteins that function together and often localize together as well. The distributions of the localized proteins were quantitated by using our recently described projected system of internal coordinates from interpolated contours (PSICIC) image analysis toolkit, leading to the identification of cellular regions that are over- or under-enriched in localized proteins and of potential differences in the mechanisms that target proteins to different subcellular destinations. The Caulobacter localizome data thus represent a resource for studying both global properties of protein localization and specific protein functions, whereas the localization analysis pipeline is a methodological resource that can be readily applied to other systems.
Solving the structure of macromolecular complexes using transmission electron microscopy can be an arduous task. Many of the steps in this process rely strongly on the aid of pre-existing structural knowledge, and are greatly complicated when this information is unavailable. Here, we present two software tools meant to facilitate particle picking, an early stage in the single-particle processing of unknown macromolecules. The first tool, DoG Picker, is an efficient and reasonably general, particle picker based on the Difference of Gaussians (DoG) image transform. It can function alone, as a reference-free particle picker with the unique ability to sort particles based on size, or it can also be used as a way to bootstrap the creation of templates or training datasets for other particle pickers. The second tool is TiltPicker, an interactive graphical interface application designed to streamline the selection of particle pairs from tilted-pair datasets. In many respects, TiltPicker is a re-implementation of the SPIDER WEB tilted-particle picker, but built on modern computer frameworks making it easier to deploy and maintain. The TiltPicker program also includes several useful new features beyond those of its predecessor.
The use of cryoEM and three-dimensional image reconstruction is becoming increasingly common. Our vision for this technique is to provide a straightforward manner in which users can proceed from raw data to a reliable 3D reconstruction through a pipeline that both facilitates management of the processing steps and makes the results at each step more transparent. Tightly integrated with a relational SQL database, Appion is a modular and transparent pipeline that extends existing software applications and procedures. The user manages and controls the software modules via web-based forms, and all results are similarly available using web-based viewers directly linked to the underlying database, enabling even naive users to quickly deduce the quality of their results. The Appion API was designed with the principle that applications should be compatible with a broad range of specimens and that libraries and routines are modular and extensible. Presented here is a description of the design and architecture of the working Appion pipeline prototype and some results of its use.
Proteins are fragile molecules that often require great care during purification to ensure that they remain intact and fully active. Removal of proteins from the cellular environment subjects them to a variety of conditions and processes that can lead to loss of activity or alteration of structure. If the protein of interest is lost or inactivated during the course of any procedure, the determination of the reason for this loss can often suggest a simple solution. Thus, if possible, it should be examined whether the loss of activity is accompanied by loss of the protein or changes in its structure or whether the protein remains but is now inactive. Protein solutions should not be exposed to extremes of pH, high temperatures, organic solvents, or any other condition that might promote denaturation. Likewise, if a protein solution is stored for extended periods in an unfrozen state, bacterial and fungal growth can become a problem.
This chapter discusses the type II restriction endonucleases—NotI and SfiI—that require an octanucleotide recognition sequence. These two endonucleases cleave deoxyribonucleic acid (DNA) less frequently than conventional tetranucleotide-, pentanucleotide-, and hexanucleotide-recognizing restriction endonucleases. On an average, NotI and SfiI will cleave DNA only once every 64,000 nucleotides. With the advent of physical methods that can separate very large DNA molecules, NotI and SfiI have become useful analytical reagents for molecular biologists. The chapter discusses several assay methods for NotI and SfiI, including agarose gel electrophoresis assay, assay for nonspecific endonuclease, and assay for exonuclease contamination. The chapter also discusses the purification and properties of NotI and SfiI. SfiI and NotI are classified as type II restriction endonucleases, because cleavage occurs at predictable nucleotide locations on both strands of the DNA molecule. Both have a strict requirement for magnesium ion, a feature common to all restriction endonuclease types, but unlike types I and III neither requires adenosine triphosphate or S-adenosylmethionine for activity.
We present a new reconstruction method that takes advantage of the fact that many biological macromolecular assemblies show a preferred orientation with respect to the plane of the specimen grid in the electron microscopic preparation. From one micrograph taken of such a specimen tilted by a large angle, a conical tilt series with random azimuthal angles can be extracted and used for a three-dimensional reconstruction. Our technique allows the determination of the molecular structure under low-dose conditions, which are not achievable with reconstruction methods that use conventional tilt series. The reconstruction method combines a number of existing image processing techniques with a newly developed weighted back-projection algorithm designed for three-dimensional reconstruction from projections taken with arbitrary projecting directions. The method is described as it was applied to the three-dimensional reconstruction of the structure of the 50S ribosomal subunit of Escherichia coli (E. coli).
The SPIDER system has evolved into a comprehensive tool set for image processing, making use of modern graphics interfacing in the VMS and UNIX environment. SPIDER and WEB handle the complementary tasks of batch processing and visualization of the results. The emphasis of the SPIDER system remains in the area of single particle averaging and reconstruction, although a variety of other application areas have been added. Novel features are a suite of operations relating to the determination, modeling, and correction of the contrast transfer function and the availability of the entire documentation in hypertext format.
One of the aims of modern microscopy is to quantify two-, three-, or even four-dimensional phenomena in biology, medicine, and material sciences. The requirements imposed on software by such data processing are exemplified by the design considerations of the IMAGIC-5 software system. This system includes facilities for multivariate statistical analysis of large data sets, for correlation averaging of two-dimensional crystals, and for three-dimensional reconstruction of macromolecular structures. The molecules may be arranged as two-dimensional crystals, as helices, or as single particles with arbitrary pointgroup symmetry. IMAGIC's novel angular reconstitution approach allows for the rapid determination of three-dimensional structures of uncrystallized molecules to high resolution. The general organization, user interaction strategy, file structure, and extendibility of IMAGIC are discussed and illustrated with some practical examples.
Restriction endonucleases are enzymes which recognize short DNA sequences and cleave the DNA in both strands. Depending on the enzymological properties different types are distinguished. Type II restriction endonucleases are homodimers which recognize short palindromic sequences 4-8 bp in length and, in the presence of Mg2+, cleave the DNA within or next to the recognition site. They are capable of non-specific binding to DNA and make use of linear diffusion to locate their target site. Binding and recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10-12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic centers. The precise mechanism of cleavage has not yet been established for any restriction endonuclease. Currently two models are discussed: the substrate-assisted catalysis mechanism and the two-metal-ion mechanism. Structural similarities identified between EcoRI, EcoRV, BamHI, PvuII and Cfr10I suggest that many type II restriction endonucleases are not only functionally but also evolutionarily related.
The rate constant for the phosphoryl transfer step in site-specific DNA cleavage by EcoRV endonuclease has been determined as a function of pH and identity of the required divalent metal ion cofactor, for both wild-type and T93A mutant enzymes. These measurements show bell-shaped pH-rate curves for each enzyme in the presence of Mg2+ as a cofactor, indicating general base catalysis for the nucleophilic attack of hydroxide ion on the scissile phosphate, and general acid catalysis for protonation of the leaving 3'-O anion. The kinetic data support a model for phosphoryl transfer based on wild-type and T93A cocrystal structures, in which the ionizations of two distinct metal-ligated waters respectively generate the attacking hydroxide ion and the proton for donation to the leaving group. The model concurs with recent observations of two metal ions bound in the active sites of the type II restriction endonucleases BamHI and BglI, suggesting the possibility of a similar catalytic mechanism functioning in many or all members of this enzyme family.
Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, an essential substrate for fatty acid biosynthesis and a potent inhibitor of fatty acid oxidation. Here, we provide evidence that glutamate may be a physiologically relevant activator of ACC. Glutamate induced the activation of both major isoforms of ACC, prepared from rat liver, heart, or white adipose tissue. In agreement with previous studies, a type 2A protein phosphatase contributed to the effects of glutamate on ACC. However, the protein phosphatase inhibitor microcystin LR did not abolish the effects of glutamate on ACC activity. Moreover, glutamate directly activated purified preparations of ACC when protein phosphatase activity was excluded. Phosphatase-independent ACC activation by glutamate was also reflected by polymerization of the enzyme as judged by size-exclusion chromatography. The sensitivity of ACC to direct activation by glutamate was diminished by treatment in vitro with AMP-activated protein kinase or cAMP-dependent protein kinase or by beta-adrenergic stimulation of intact adipose tissue. We conclude that glutamate, an abundant intracellular amino acid, induces ACC activation through complementary actions as a phosphatase activator and as a direct allosteric ligand for dephosphorylated ACC. This study supports the general hypothesis that amino acids fulfill important roles as signal molecules as well as intermediates in carbon and nitrogen metabolism.
The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.
The FindEM particle picking program was tested on the publicly available keyhole limpet hemocyanin (KLH) dataset, and the results were submitted for the "bakeoff" contest at the recent particle picking workshop (Zhu et al., 2003b). Two alternative ways of using the program are demonstrated and the results are compared. The first of these approximates exhaustive projection matching with a full set of expected views, which need to be known. This could correspond to the task of extending a known structure to higher resolution, for which many 1000's of additional images are required. The second procedure illustrates use of multivariate statistical analysis (MSA) to filter a preliminary set of candidate particles containing a high proportion of false particles. This set was generated using the FindEM program to search with one template that crudely represents the expected views. Classification of the resultant set of candidate particles then allows the desired classes to be selected while the rest can be ignored. This approach requires no prior information of the structure and is suitable for the initial investigation of an unknown structure--the class averages indicate the symmetry and oligomeric state of the particles. Potential improvements in speed and accuracy are discussed.