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Single turnover DNA cleavage rate constants (kobs) of 1 nM 32P labeled 18–1 DNA, 1 μM SgrAI enzyme dimer mutant and varied concentration of unlabeled PC DNA.
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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 secondar...
Citations
... WT SgrAI enzyme was prepared as previously described (20). The expression vector for the K242A mutant of SgrAI was prepared using a commercial source, but because this mutant form had more limited solubility, a second mutation was introduced, L336K. ...
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.
... However, the R state remains energetically accessible, even when SgrAI is bound to secondary sequences, which enables filamentation nucleated by SgrAI bound to primary sites and leads to a cleavage cascade. Hence, according to this model, cleavage of secondary sequences will occur appreciably only in the presence of SgrAI bound to primary sequences, which is observed experimentally [25,43,46,59]. ...
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 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). The filamentous state preferentially stabilizes the activated conformation of the enzyme; hence, SgrAI in the filament is activated for DNA cleavage (Polley et al. 2019). ...
... Hence, the origin of the differential DNA cleavage activity on the two types of sequences, primary and secondary, may originate in DNA structure and energy with filamentation serving as the means to detect this energy. In the filament, SgrAI cleaves both types of sites rapidly resulting in a 200fold acceleration in the case of primary sites, 1000-fold in the case of secondary (Shah et al. 2015). The expansion of DNA cleavage activity from primary to primary and secondary increases the number of cleavage sequences from 3 to 17. ...
... Redox stress, high hydrogen peroxide levels, heat shock, low pH, and site-specific phosphorylation induce filamentation. Helical reconstruction and cryo-EM (3.5 Å resolution) shows left handed helix with~T he enzyme is 200-1000× activated in the filament Shah et al. 2015). ...
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.
... 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). ...
... Structural changes proximal to secondary site substitutions within filamentous SgrAI provide insights into sequence specificity Upon filamentation, SgrAI exhibits an apparent expansion in DNA cleavage sequence specificity. In the nonfilamentous low-activity form, SgrAI cleaves only its primary site sequences (CR|CCGGYG, R = A and G; and Y = C and T, and | indicates site of cleavage) but not its secondary site sequences (CC| CCGGYG, DR|CCGGYG, D = T, A, and G), despite the fact that both types of sites are bound by the enzyme with high affinity (12,17). Filamentation of DNA-bound SgrAI stabilizes a change in enzyme conformation, which activates DNA cleavage activity on both primary and secondary sites (12,16). ...
... The T-state and R-state models of Figure 6 can also be used to understand the expansion of DNA cleavage sequence specificity (i.e., the secondary site cleavage activity of SgrAI). If the T state is more favored when secondary site sequences are bound to SgrAI than when primary site sequences are bound, then SgrAI bound to secondary site DNA will be less inclined to filament, and DNA cleavage of secondary sites will be low, which is what is observed experimentally (12,17). Filamentation can be driven by increasing Filamentation-induced activation of SgrAI the concentration of R states, which occurs when SgrAI bound to primary sites are also present; this increase in R-state species will drive the equilibrium of SgrAI bound to secondary site sequences to the right also, thereby inducing secondary site cleavage by SgrAI, which is again what is observed experimentally (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.
... Incorporation of 3 S and 5 S linkages into oligonucleotides is possible by standard solid phase oligonucleotide synthesis using appropriate phosphoramidite monomers ( Figure 1C (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)) and the hybridization behavior as well as the conformational properties of such oligonucleotides have been reported (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). 3 S and 5 S thiophosphates have also been applied in mechanistic studies with enzymes Downloaded from https://academic.oup.com/nar/article-abstract/48/1/63/5637591 by guest on 25 March 2020 and ribozymes (39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50) and in site selective chemical strand cleavage (31,32,34,35). In one study investigating the 5 S modification in RNase H activating gapmers a hitherto unexplained discrepancy between in vitro and in vivo activity was observed (37,38). ...
The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.
... 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). The filamentous state preferentially stabilizes the activated conformation of the enzyme; hence, SgrAI in the filament is activated for DNA cleavage (Polley et al. 2019). ...
... Hence, the origin of the differential DNA cleavage activity on the two types of sequences, primary and secondary, may originate in DNA structure and energy with filamentation serving as the means to detect this energy. In the filament, SgrAI cleaves both types of sites rapidly resulting in a 200fold acceleration in the case of primary sites, 1000-fold in the case of secondary (Shah et al. 2015). The expansion of DNA cleavage activity from primary to primary and secondary increases the number of cleavage sequences from 3 to 17. ...
... Redox stress, high hydrogen peroxide levels, heat shock, low pH, and site-specific phosphorylation induce filamentation. Helical reconstruction and cryo-EM (3.5 Å resolution) shows left handed helix with~T he enzyme is 200-1000× activated in the filament Shah et al. 2015). ...
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.
... 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 . The filamentous state preferentially stabilizes the activated conformation of the enzyme, hence SgrAI in the filament is activated for DNA cleavage 39 . ...
... Hence the origin of the differential DNA cleavage activity on the two types of sequences, primary and secondary, may originate in DNA structure and energy with filamentation serving as the means to detect this energy. In the filament, SgrAI cleaves both types of sites rapidly resulting in a 200 fold acceleration in the case of primary sites, 1000 fold in the case of secondary 38 . The expansion of DNA cleavage activity from primary to primary and secondary increases the number of cleavage sequences from 3 to 17. ...
... In only a few enzymes does the substrate specificity change when in the filament form. SgrAI, a sequence specific endonuclease, cleaves 17 different 8 bp DNA sequences in the filamentous site, but only 3 in the non-filamentous state 17,35,38 . IRE1 may also have expanded substrate specificity when filamentous, cleaving other RNAs in addition to its canonical RNA substrate 240 . ...
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.
... Its primary recognition sequence in double-stranded DNA (CR|CCGGYG, where R = A or G and Y = C or T; | denotes cleavage site) can both be cleaved and serve as an allosteric activator. When activated, SgrAI cleaves primary sites in DNA over 200-fold faster, but also alters its sequence specificity to include an additional 14 DNA sequences, known as secondary sites (CCCCGGYG or DRCCGGYG, where D = A, G, or T, and underlined nucleotides are those that differ in secondary sites from primary) (Bitinaite and Schildkraut, 2002;Shah et al., 2015). In the absence of DNA, SgrAI is a homodimer composed of two 37-kDa chains, each with a single active site . ...
... SgrAI DBD bound to a secondary-site DNA disfavors the activated conformation, and hence ROO filament formation by SgrAI bound to a secondary site does not appreciably occur ( Figure 1B). This explains why cleavage of secondary-site sequences is negligible unless a primary site is present-in sufficient concentration or on the same contiguous DNA-to promote ROO filament assembly (Bitinaite and Schildkraut, 2002;Park et al., 2010;Shah et al., 2015). Filaments formed by SgrAI bound to primary-site DNA will drive ROO filament assembly, incorporating DBD-containing secondary sites, activating SgrAI for DNA cleavage on both primary and secondary sites, and thereby altering the sequence specificity of the enzyme ( Figure 1C). ...
... To elucidate the molecular determinants of SgrAI activation, we sought to resolve the ROO filament to near-atomic resolution. We prepared ROO filaments from purified, recombinant, Histagged wild-type SgrAI (Shah et al., 2015) bound to a 40-bp oligonucleotide DNA containing a pre-cleaved primary-site sequence (PC DNA, see STAR Methods). Cryo-EM micrographs revealed a large variety of differently sized filaments, as expected for an ROO ( Figure S1A). ...
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.
... 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). ...
... Its cleavage of secondary sites occurs only under particular conditions, namely, when present on the same DNA molecule as a primary site, or alternatively, when in the presence of high concentrations of both SgrAI and primary site DNA sequences on separate DNA molecules (7,(20)(21)(22)(23). The same conditions leading to cleavage of secondary sites by SgrAI also accelerate the cleavage of primary sites by SgrAI over 200-fold (the acceleration of secondary site cleavage is approximately 1,000-fold) (7,16,20,22). Further, under these conditions, SgrAI forms the ROO filament described above, thought to stabilize the activated state of SgrAI (8). The role of this unexpected structure was not known and has been the subject of recent investigations. ...
... The single-turnover DNA cleavage rate constant under activating conditions was also measured for each SgrAI mutant. These assays include 1 M PC DNA, which activates SgrAI into forming ROO filaments with accelerated DNA cleavage activity (7,8,16). PC DNA is a precleaved 40-bp DNA containing a single primary site sequence (see Materials and Methods for the sequence). ...
... 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). ...
... Its cleavage of secondary sites occurs only under particular conditions, namely, when present on the same DNA molecule as a primary site, or alternatively, when in the presence of high concentrations of both SgrAI and primary site DNA sequences on separate DNA molecules (7,(20)(21)(22)(23). The same conditions leading to cleavage of secondary sites by SgrAI also accelerate the cleavage of primary sites by SgrAI over 200-fold (the acceleration of secondary site cleavage is approximately 1,000-fold) (7,16,20,22). Further, under these conditions, SgrAI forms the ROO filament described above, thought to stabilize the activated state of SgrAI (8). The role of this unexpected structure was not known and has been the subject of recent investigations. ...
... The single-turnover DNA cleavage rate constant under activating conditions was also measured for each SgrAI mutant. These assays include 1 M PC DNA, which activates SgrAI into forming ROO filaments with accelerated DNA cleavage activity (7,8,16). PC DNA is a precleaved 40-bp DNA containing a single primary site sequence (see Materials and Methods for the sequence). ...
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.
