@article {2022|2156, title = {Building Biological Relevance Into Integrative Modelling of Macromolecular Assemblies}, journal = {Frontiers in Molecular Biosciences}, volume = {9}, year = {2022}, pages = {826136}, abstract = {

Recent advances in structural biophysics and integrative modelling methods now allow us to decipher the structures of large macromolecular assemblies. Understanding the dynamics and mechanisms involved in their biological function requires rigorous integration of all available data. We have developed a complete modelling pipeline that includes analyses to extract biologically significant information by consistently combining automated and interactive human-guided steps. We illustrate this idea with two examples. First, we describe the ryanodine receptor, an ion channel that controls ion flux across the cell membrane through transitions between open and closed states. The conformational changes associated with the transitions are small compared to the considerable system size of the receptor; it is challenging to consistently track these states with the available cryo-EM structures. The second example involves homologous recombination, in which long filaments of a recombinase protein and DNA catalyse the exchange of homologous DNA strands to reliably repair DNA double-strand breaks. The nucleoprotein filament reaction intermediates in this process are short-lived and heterogeneous, making their structures particularly elusive. The pipeline we describe, which incorporates experimental and theoretical knowledge combined with state-of-the-art interactive and immersive modelling tools, can help overcome these challenges. In both examples, we point to new insights into biological processes that arise from such interdisciplinary approaches.

}, issn = {2296-889X}, doi = {10.3389/fmolb.2022.826136}, url = {https://www.frontiersin.org/article/10.3389/fmolb.2022.826136}, author = {Molza, Anne-Elisabeth and Westermaier, Yvonne and Moutte, Magali and Ducrot, Pierre and Danilowicz, Claudia and Godoy-Carter, Veronica and Prentiss, Mara and Robert, Charles H. and Marc Baaden and Pr{\'e}vost, Chantal} } @article {2019|2062, title = {The positioning of Chi sites allows the RecBCD pathway to suppress some genomic rearrangements}, journal = {Nucleic Acids Res}, volume = {47}, year = {2019}, month = {02}, pages = {1836-1846}, abstract = {

Bacterial recombinational repair of double-strand breaks often begins with creation of initiating 3\&$\#$39; single-stranded DNA (ssDNA) tails on each side of a double-strand break (DSB). Importantly, if the RecBCD pathway is followed, RecBCD creates a gap between the sequences at 3\&$\#$39; ends of the initiating strands. The gap flanks the DSB and extends at least to the nearest Chi site on each strand. Once the initiating strands form ssDNA-RecA filaments, each ssDNA-RecA filament searches for homologous double-stranded DNA (dsDNA) to use as a template for the DNA synthesis needed to fill the gap created by RecBCD. Our experimental results show that the DNA synthesis requires formation of a heteroduplex dsDNA that pairs \>20 contiguous bases in the initiating strand with sequence matched bases in a strand from the original dsDNA. To trigger synthesis, the heteroduplex must be near the 3\&$\#$39; end of the initiating strand. Those experimentally determined requirements for synthesis combined with the Chi site dependence of the function of RecBCD and the distribution of Chi sites in bacterial genomes could allow the RecBCD pathway to avoid some genomic rearrangements arising from directly induced DSBs; however, the same three factors could promote other rearrangements.

}, doi = {10.1093/nar/gky1252}, author = {Li, Chastity and Danilowicz, Claudia and Tashjian, Tommy F and Godoy, Veronica G and Chantal Pr{\'e}vost and Prentiss, Mara} } @article {2019|2063, title = {Residues in the fingers domain of the translesion DNA polymerase DinB enable its unique participation in error-prone double-strand break repair}, journal = {J Biol Chem}, volume = {294}, year = {2019}, month = {May}, pages = {7588-7600}, abstract = {

The evolutionarily conserved Escherichia coli translesion DNA polymerase IV (DinB) is one of three enzymes that can bypass potentially deadly DNA lesions on the template strand during DNA replication. Remarkably, however, DinB is the only known translesion DNA polymerase active in RecA-mediated strand exchange during error-prone double-strand break repair. In this process, a single-stranded DNA (ssDNA)-RecA nucleoprotein filament invades homologous dsDNA, pairing the ssDNA with the complementary strand in the dsDNA. When exchange reaches the 3\&$\#$39; end of the ssDNA, a DNA polymerase can add nucleotides onto the end, using one strand of dsDNA as a template and displacing the other. It is unknown what makes DinB uniquely capable of participating in this reaction. To explore this topic, we performed molecular modeling of DinB\&$\#$39;s interactions with the RecA filament during strand exchange, identifying key contacts made with residues in the DinB fingers domain. These residues are highly conserved in DinB, but not in other translesion DNA polymerases. Using a novel FRET-based assay, we found that DinB variants with mutations in these conserved residues are less effective at stabilizing RecA-mediated strand exchange than native DinB. Furthermore, these variants are specifically deficient in strand displacement in the absence of RecA filament. We propose that the amino acid patch of highly conserved residues in DinB-like proteins provides a mechanistic explanation for DinB\&$\#$39;s function in strand exchange and improves our understanding of recombination by providing evidence that RecA plays a role in facilitating DinB\&$\#$39;s activity during strand exchange.

}, keywords = {DinB, DNA damage, DNA polymerase, DNA polymerase IV, DNA repair, DNA synthesis, homologous recombination, RecA}, doi = {10.1074/jbc.RA118.006233}, author = {Tashjian, Tommy F and Danilowicz, Claudia and Molza, Anne-Elizabeth and Nguyen, Brian H and Chantal Pr{\'e}vost and Prentiss, Mara and Godoy, Veronica G} } @article {2019|2064, title = {Slow extension of the invading DNA strand in a D-loop formed by RecA-mediated homologous recombination may enhance recognition of DNA homology}, journal = {J Biol Chem}, volume = {294}, year = {2019}, month = {May}, pages = {8606-8616}, abstract = {

DNA recombination resulting from RecA-mediated strand exchange aided by RecBCD proteins often enables accurate repair of DNA double-strand breaks. However, the process of recombinational repair between short DNA regions of accidental similarity can lead to fatal genomic rearrangements. Previous studies have probed how effectively RecA discriminates against interactions involving a short similar sequence that is embedded in otherwise dissimilar sequences but have not yielded fully conclusive results. Here, we present results of in vitro experiments with fluorescent probes strategically located on the interacting DNA fragments used for recombination. Our findings suggest that DNA synthesis increases the stability of the recombination products. Fluorescence measurements can also probe the homology dependence of the extension of invading DNA strands in D-loops formed by RecA-mediated strand exchange. We examined the slow extension of the invading strand in a D-loop by DNA polymerase (Pol) IV and the more rapid extension by DNA polymerase LF-Bsu We found that when DNA Pol IV extends the invading strand in a D-loop formed by RecA-mediated strand exchange, the extension afforded by 82 bp of homology is significantly longer than the extension on 50 bp of homology. In contrast, the extension of the invading strand in D-loops by DNA LF-Bsu Pol is similar for intermediates with \≥50 bp of homology. These results suggest that fatal genomic rearrangements due to the recombination of small regions of accidental homology may be reduced if RecA-mediated strand exchange is immediately followed by DNA synthesis by a slow polymerase.

}, keywords = {cooperativity, DNA damage, DNA polymerase, DNA recombination, double-strand break (DSB), fluorescence resonance energy transfer (FRET), heteroduplex formation, molecular dynamics, RecA, strand displacement synthesis}, doi = {10.1074/jbc.RA119.007554}, author = {Lu, Daniel and Danilowicz, Claudia and Tashjian, Tommy F and Chantal Pr{\'e}vost and Godoy, Veronica G and Prentiss, Mara} } @article {2019|2065, title = {Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination}, journal = {Nucleic Acids Res}, volume = {47}, year = {2019}, month = {Sep}, pages = {7798-7808}, abstract = {

Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.

}, doi = {10.1093/nar/gkz667}, author = {Boyer, Benjamin and Danilowicz, Claudia and Prentiss, Mara and Chantal Pr{\'e}vost} } @article {2017|2022, title = {ATP hydrolysis provides functions that promote rejection of pairings between different copies of long repeated sequences}, journal = {Nucleic Acids Res}, volume = {45}, year = {2017}, pages = {8448-8462}, abstract = {

During DNA recombination and repair, RecA family proteins must promote rapid joining of homologous DNA. Repeated sequences with \>100 base pair lengths occupy more than 1\% of bacterial genomes; however, commitment to strand exchange was believed to occur after testing ~20-30 bp. If that were true, pairings between different copies of long repeated sequences would usually become irreversible. Our experiments reveal that in the presence of ATP hydrolysis even 75 bp sequence-matched strand exchange products remain quite reversible. Experiments also indicate that when ATP hydrolysis is present, flanking heterologous dsDNA regions increase the reversibility of sequence matched strand exchange products with lengths up to ~75 bp. Results of molecular dynamics simulations provide insight into how ATP hydrolysis destabilizes strand exchange products. These results inspired a model that shows how pairings between long repeated sequences could be efficiently rejected even though most homologous pairings form irreversible products.

}, doi = {10.1093/nar/gkx582}, author = {Danilowicz, Claudia and Hermans, Laura and Coljee, Vincent and Chantal Pr{\'e}vost and Prentiss, Mara} } @article {2015|1731, title = {Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure}, journal = {Nucleic Acids Res.}, volume = {43}, year = {2015}, month = {dec}, pages = {10251{\textendash}63}, abstract = {

RecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results.

}, doi = {10.1093/nar/gkv883}, author = {Yang, Darren and Boyer, Benjamin and Chantal Pr{\'e}vost and Danilowicz, Claudia and Prentiss, Mara} } @article {2015|1732, title = {The poor homology stringency in the heteroduplex allows strand exchange to incorporate desirable mismatches without sacrificing recognition in vivo}, journal = {Nucleic Acids Res.}, volume = {43}, year = {2015}, month = {jul}, pages = {6473{\textendash}85}, abstract = {

RecA family proteins are responsible for homology search and strand exchange. In bacteria, homology search begins after RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site, forming the presynaptic filament. Once the filament is formed, it interrogates double-stranded DNA (dsDNA). During the interrogation, bases in the dsDNA attempt to form Watson-Crick bonds with the corresponding bases in the initiating strand. Mismatch dependent instability in the base pairing in the heteroduplex strand exchange product could provide stringent recognition; however, we present experimental and theoretical results suggesting that the heteroduplex stability is insensitive to mismatches. We also present data suggesting that an initial homology test of 8 contiguous bases rejects most interactions containing more than 1/8 mismatches without forming a detectable 20 bp product. We propose that, in vivo, the sparsity of accidental sequence matches allows an initial 8 bp test to rapidly reject almost all non-homologous sequences. We speculate that once the initial test is passed, the mismatch insensitive binding in the heteroduplex allows short mismatched regions to be incorporated in otherwise homologous strand exchange products even though sequences with less homology are eventually rejected.

}, doi = {10.1093/nar/gkv610}, author = {Danilowicz, Claudia and Yang, Darren and Kelley, Craig and Chantal Pr{\'e}vost and Prentiss, Mara} } @article {2015|1641, title = {Structure/function relationships in RecA protein-mediated homology recognition and strand exchange}, journal = {Crit. Rev. Biochem. Mol. Biol.}, volume = {50}, year = {2015}, pages = {453{\textendash}76}, abstract = {RecA family proteins include RecA, Rad51, and Dmc1. These recombinases are responsible for homology search and strand exchange. Homology search and strand exchange occur during double-strand break repair and in eukaryotes during meiotic recombination. In bacteria, homology search begins when RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site to form the presynaptic filament. The filament is a right-handed helix, where the initiating strand is bound deep within the filament. Once the presynaptic filament is formed, it interrogates nearby double-stranded DNA (dsDNA) to find a homologous sequence; therefore, we provide a detailed discussion of structural features of the presynaptic filament that play important functional roles. The discussion includes many diagrams showing multiple filament turns. These diagrams illustrate interactions that are not evident in single turn structures. The first dsDNA interactions with the presynaptic filament are insensitive to mismatches. The mismatch insensitive interactions lead to dsDNA deformation that triggers a homology testing process governed by kinetics. The first homology test involves {\^a}ˆ{\textonequarter}8 bases. Almost all interactions are rejected by this initial rapid test, leading to a new cycle of homology testing. Interactions that pass the initial rapid test proceed to a slower testing stage. That slower stage induces nonhomologous dsDNA to reverse strand exchange and begin a new cycle of homology testing. In contrast, homologous dsDNA continues to extend the heteroduplex strand-exchange product until ATP hydrolysis makes strand exchange irreversible.}, keywords = {Double-strand break repair, meiosis, meiotic recombination, Rad51, recombinase}, doi = {10.3109/10409238.2015.1092943}, author = {Prentiss, Mara and Chantal Pr{\'e}vost and Danilowicz, Claudia} }