[Show abstract][Hide abstract] ABSTRACT: Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.
Full-text · Article · Jan 2016 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.
Full-text · Article · Dec 2015 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These
enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage
even in complete genomes. Although a large number of homing endonucleases have been identified, the landscape of possible
target sequences is still very limited to cover the complexity of the whole eukaryotic genome. Therefore, the finding and
molecular analysis of homing endonucleases identified but not yet characterized may widen the landscape of possible target
sequences. The previous characterization of protein-DNA interaction before the engineering of new homing endonucleases is
essential for further enzyme modification. Here we report the crystal structure of I-CvuI in complex with its target DNA and
with the target DNA of I-CreI, a homologue enzyme widely used in genome engineering. To characterize the enzyme cleavage mechanism,
we have solved the I-CvuI DNA structures in the presence of non-catalytic (Ca2+) and catalytic ions (Mg2+). We have also analyzed the metal dependence of DNA cleavage using Mg2+ ions at different concentrations ranging from non-cleavable to cleavable concentrations obtained from in vitro cleavage experiments. The structure of I-CvuI homing endonuclease expands the current repertoire for engineering custom specificities,
both by itself as a new scaffold alone and in hybrid constructs with other related homing endonucleases or other DNA-binding
No preview · Article · Sep 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: From crystallography, the technique mostly used to study the structure of matter, the field mutated into structural biology, has mutated in life sciences into structural biology, which has been developed as an essential and rather successful area of research to fully understand the workings of cellular pathways. The application of physical approaches to biological systems has been crucial to comprehend the structure and function of the biological components of living organisms. In this assay the author walks the reader through the last century, which has witnessed how this life sciences research area was born and moved towards larger assemblies in the core of crucial biological problems. The influence of research in physics, biochemistry and molecular biology has been key in the successes and large body of seminal results obtained by structural biologists. The author proposes that the future of this area implies the integration of its results at the cellular level apart of using more quantitative approaches to describe biological processes.
[Show abstract][Hide abstract] ABSTRACT: The presence of germline mutations affecting the MYC-associated protein X (MAX) gene has recently been identified as one of the now 11 major genetic predisposition factors for the development of hereditary pheochromocytoma and/or paraganglioma. Little is known regarding how missense variants of unknown significance (VUS) in MAX affect its pivotal role in the regulation of the MYC/MAX/MXD axis. In the present study, we propose a consensus computational prediction based on five "state-of-the-art" algorithms. We also describe a PC12-based functional assay to assess the effects that 12 MAX VUS may have on MYC's E-box transcriptional activation. For all but two of these 12 VUS, the functional assay and the consensus computational prediction gave consistent results; we classified seven variants as pathogenic and three as nonpathogenic. The introduction of wild-type MAX cDNA into PC12 cells significantly decreased MYC's ability to bind to canonical E-boxes, while pathogenic MAX proteins were not able to fully repress MYC activity. Further clinical and molecular evaluation of variant carriers corroborated the results obtained with our functional assessment. In the absence of clear heritability, clinical information, and molecular data, consensus computational predictions and functional models are able to correctly classify VUS affecting MAX.
A functional assay assesses the effects of MAX VUS over MYC transcriptional activity. A consensus computational prediction and the functional assay show high concordance. Variant carriers' clinical and molecular data support the functional assessment.
No preview · Article · Jun 2015 · Journal of Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: The intrinsically disordered protein p15 PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15 PAF –PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15 PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15 PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15 PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15 PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15 PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.
[Show abstract][Hide abstract] ABSTRACT: The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We observed the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site. This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals during catalysis.
[Show abstract][Hide abstract] ABSTRACT: chTOG is a conserved microtubule polymerase that catalyses the addition of tubulin dimers to promote microtubule growth. chTOG interacts with TACC3, a member of the transforming acidic coiled-coil (TACC) family. Here we analyse their association using the Xenopus homologues, XTACC3 (TACC3) and XMAP215 (chTOG), dissecting the mechanism by which their interaction promotes microtubule elongation during spindle assembly. Using SAXS, we show that the TACC domain (TD) is an elongated structure that mediates the interaction with the C terminus of XMAP215. Our data suggest that one TD and two XMAP215 molecules associate to form a four-helix coiled-coil complex. A hybrid methods approach was used to define the precise regions of the TACC heptad repeat and the XMAP215 C terminus required for assembly and functioning of the complex. We show that XTACC3 can induce the recruitment of larger amounts of XMAP215 by increasing its local concentration, thereby promoting efficient microtubule elongation during mitosis.
[Show abstract][Hide abstract] ABSTRACT: DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endo-genous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing 'toolkits' for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.
[Show abstract][Hide abstract] ABSTRACT: Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in chromatin dynamics, including DNA replication and repair, transcription and chromosome segregation. The two members of the family reported in humans, namely TLK1 and TLK2, localize to the cell nucleus and are capable of forming homo- or hetero-oligomers by themselves. To characterize the role of TLK2, its C-terminal kinase domain was cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4122 and cubic P213. The latter produced the best diffracting crystal (3.4 Å resolution using synchrotron radiation), with unit-cell parameters a = b = c = 126.05 Å, α = β = γ = 90°. The asymmetric unit contained one protein molecule, with a Matthews coefficient of 4.59 Å(3) Da(-1) and a solvent content of 73.23%.
[Show abstract][Hide abstract] ABSTRACT: A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only
one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate
one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable.
Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable
diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The
analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a
TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report
that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable
activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for
more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.
Full-text · Article · Feb 2014 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Homing endonucleases are highly specific DNA-cleaving enzymes that recognize long stretches of DNA. The engineering of these enzymes provides novel instruments for genome modification in a wide range of fields, including gene targeting, by inducing specific double-strand breaks. I-CvuI is a homing endonuclease from the green alga Chlorella vulgaris. This enzyme was purified after overexpression in Escherichia coli. Crystallization experiments of I-CvuI in complex with its DNA target in the presence of Mg(2+) yielded crystals suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 62.83, b = 83.56, c = 94.40 Å. The self-rotation function and the Matthews coefficient suggested the presence of one protein-DNA complex per asymmetric unit. The crystals diffracted to a resolution limit of 1.9 Å using synchrotron radiation.
[Show abstract][Hide abstract] ABSTRACT: Synopsis: The homing endonuclease I-CvuI from C. vulgaris has been expressed, purified and crystallized in complex with its target DNA. Preliminary X-ray diffraction analysis is reported. ARTICLE IN PRESS – Acta Cryst. F Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease of Chlorella vulgaris I-CvuI in complex with its target DNA How to cite your article in press Your article has not yet been assigned page numbers, but may be cited using the doi: You will be sent the full citation when your article is published and also given instructions on how to download an electronic reprint of your article. Proof instructions Proof corrections should be returned by 19 January 2014. After this period, the Editors reserve the right to publish your article with only the Managing Editor's corrections. Please (1) Read these proofs and assess whether any corrections are necessary. (2) Check that any technical editing queries highlighted in bold underlined text have been answered. (3) Send corrections by e-mail to email@example.com. Please describe corrections using plain text, where possible, giving the line numbers indicated in the proof. Please do not make corrections to the pdf file electronically and please do not return the pdf file. If no corrections are required please let us know. If you wish to make your article open access or purchase printed offprints, please complete the attached order form and return it by e-mail as soon as possible. Thumbnail image for contents page Files: f/fw5442/fw5442.3d f/fw5442/fw5442.sgml FW5442 XC IU-1414/43(16)1 1412/29(16)1 () Homing endonucleases are highly specific DNA-cleaving enzymes that recognize long stretches of DNA. The engineering of these enzymes provides novel instruments for genome modification in a wide range of fields, including gene targeting by inducing specific double-strand breaks. I-CvuI is a homing endonuclease from the green alga Chlorella vulgaris. This enzyme was purified after overexpression in Escherichia coli. Crystallization experiments of I-CvuI in complex with its DNA target in the presence of Mg 2+ yielded crystals suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 62.83, b = 83.56, c = 94.40 Å , = = = 90 . The self-rotation function and the Matthews coefficient suggested the presence of one protein–DNA complex per asymmetric unit. The crystals diffracted to a resolution limit of 1.9 Å using synchrotron radiation.
[Show abstract][Hide abstract] ABSTRACT: Different genome-editing strategies have fuelled the development of new DNA-targeting molecular tools allowing precise gene modifications. Here, the expression, purification, crystallization and preliminary X-ray diffraction of BurrH, a novel DNA-binding protein from Burkholderia rhizoxinica, are reported. Crystallization experiments of BurrH in its apo form and in complex with its target DNA yielded crystals suitable for X-ray diffraction analysis. The crystals of the apo form belonged to the primitive hexagonal space group P3(1) or its enantiomorph P3(2), with unit-cell parameters a = b = 73.28, c = 268.02 angstrom, alpha = beta = 90, gamma = 120 degrees. The BurrH-DNA complex crystallized in the monoclinic space group P2(1), with unit-cell parameters a = 70.15, b = 95.83, c = 76.41 angstrom, alpha = gamma = 90, beta = 109.51 degrees. The self-rotation function and the Matthews coefficient suggested the presence of two protein molecules per asymmetric unit in the apo crystals and one protein-DNA complex in the monoclinic crystals. The crystals diffracted to resolution limits of 2.21 and 2.65 angstrom, respectively, using synchrotron radiation.
Full-text · Article · Jan 2014 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
[Show abstract][Hide abstract] ABSTRACT: Transcription activator-like effectors contain a DNA-binding domain organized in tandem repeats. The repeats include two adjacent residues known as the repeat variable di-residue, which recognize a single base pair, establishing a direct code between the dipeptides and the target DNA. This feature suggests this scaffold as an excellent candidate to generate new protein-DNA specificities for biotechnological applications. Here, the crystal structure of AvrBs3 (residues 152-895, molecular mass 82 kDa) in complex with its target DNA sequence is presented, revealing a new mode of interaction with the initial thymine of the target sequence, together with an analysis of both the binding specificity and the thermodynamic properties of AvrBs3. This study quantifies the affinity and the specificity between AvrBs3 and its target DNA. Moreover, in vitro and in vivo analyses reveal that AvrBs3 does not show a strict nucleotide-binding preference for the nucleotide at the zero position of the DNA, widening the number of possible sequences that could be targeted by this scaffold.