Biology subjects

Pages, V.

Publications and source records attributed to Pages, V..

11 recordsLinked to original sources

bioRxiv2024

Rad51 determines pathway usage in post-replication repair

Stalled replication forks can be processed by several distinct mechanisms collectively called post-replication repair which includes homologous recombination, fork regression, and translesion DNA synthesis. However, the regulation of the usage between these pathways is not fully understood. The Rad51 protein plays a pivotal role in maintaining genomic stability through its roles in HR and in protecting stalled replication forks from degradation. We report the isolation of separation-of-function mutations in Saccharomyces cerevisiae Rad51 that retain their recombination function but display a defect in fork protection leading to a shift in post-replication repair pathway usage from HR to alternate pathways including mutagenic translesion synthesis. Rad51-E135D and Rad51-K305N show normal in vivo and in vitro recombination despite changes in their DNA binding profiles, in particular to dsDNA, with a resulting effect on their ATPase activities. The mutants lead to a defect in Rad51 recruitment to stalled forks in vivo as well as a defect in the protection of dsDNA from degradation by Dna2-Sgs1 and Exo1 in vitro. A high-resolution cryo-electron microscopy structure of the Rad51-ssDNA filament at 2.4 [A] resolution provides a structural basis for a mechanistic understanding of the mutant phenotypes. Together, the evidence suggests a model in which Rad51 binding to duplex DNA is critical to control pathway usage at stalled replication forks.

molecular biology↗

bioRxiv2024

A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS

Processivity clamps mediate polymerase switching for translesion synthesis (TLS). All three E. coli TLS polymerases interact with the {beta}2 processivity clamp through a conserved clamp-binding motif (CBM), which is indispensable for TLS. Notably, Pol IV also makes a unique secondary contact with the clamp through non-CBM residues. However, the role of this "rim contact" in Pol IV-mediated TLS remains poorly understood. Here we show that the rim contact is critical for TLS past strong replication blocks. In in vitro reconstituted Pol IV-mediated TLS, ablating the rim contact compromises TLS past 3-methyl dA, a strong block, while barely affecting TLS past N2-furfuryl dG, a weak block. Similar observations are also made in E. coli cells bearing a single copy of these lesions in the genome. Within lesion-stalled replication forks, the rim interaction and ssDNA binding protein cooperatively poise Pol IV to better compete with Pol III for binding to a cleft through its CBM. We propose that this bipartite clamp interaction enables Pol IV to rapidly resolve lesion-stalled replication through TLS at the fork, which reduces damage induced mutagenesis.

biochemistry↗

bioRxiv2023

The RecBC complex protects the nascent DNA in the presence of single-stranded DNA gaps

Following encounter with an unrepaired DNA lesion, replication is halted and can restart downstream of the lesion leading to the formation of a single-stranded DNA (ssDNA) gap. To complete replication, this ssDNA gap is filled in by one of the two lesion tolerance pathways: the error-prone Translesion Synthesis (TLS) or the error-free Homology Directed Gap Repair (HDGR). In the present work, we evidence a new role for the RecBC complex distinct from its canonical function in homologous recombination at DNA double strand breaks. We show that upon lesion encounter RecBC (independently of its catalytic activity and of the RecD subunit) is required to protect the nascent DNA in order to promote efficient lesion bypass. In the absence of RecBC, our data indicate that the nuclease ExoI can access and degrade the nascent DNA, affecting both TLS and HDGR mechanisms. We show that the recruitment of RecBC becomes particularly important at strong blocking lesions, when post-replicatively ssDNA gaps persist and are covered by the single-stranded DNA binding protein (SSB). This protective role of RecBC is reminiscent of the role of BRCA2 in protecting the nascent DNA in human cells, highlighting once again the evolutionary conservation of DNA replication mechanisms across all living organisms. SIGNIFICANCE STATEMENTIn the presence of unrepaired lesions, stalled replication can restart ahead of the lesion creating a single stranded DNA gap. To preserve genome stability, this gap needs to be later filled in by one of the two lesion tolerance pathways, Translesion Synthesis or Homology Directed Gap Repair. In this study, we unveil a role for the RecBC complex in protecting the nascent DNA blocked at the lesion site from degradation by the ExoI nuclease. This protection is essential for an efficient repair of the single-stranded DNA gap. In the absence of RecBC, both lesion tolerance pathways are affected and genome stability is compromised.

molecular biology↗

bioRxiv2023

Cells limit mutagenesis by dealing with DNA lesions behind the fork

DNA lesions are a threat to genome stability. To cope with them during DNA replication, cells have evolved lesion bypass mechanisms: Translesion Synthesis (TLS), which allows the cell to insert a nucleotide directly opposite the lesion, with the risk of introducing a mutation, and error-free Damage Avoidance (DA), which uses homologous recombination to retrieve the genetic information from the sister chromatid. In this study, we investigate the timing of lesion bypass and its implications for the accuracy of the process. Our findings reveal that DNA polymerase {eta} can bypass common, UV-induced TT-cyclobutane pyrimidine dimers at the fork, immediately after encountering the blocking lesion. In contrast, TLS at TT(6-4) photoproducts and bulky G-AAF adducts, mediated by Rev1 and Pol {zeta}, takes place behind the fork, at post-replicative gaps that are generated downstream of the lesion after repriming. We show that in this latter situation, TLS competes with the DA pathway, thus reducing overall mutagenicity of damage bypass. Additionally, our study demonstrates that Exo1 nuclease influences the balance between TLS and DA by modulating the size of the post-replicative gaps.

molecular biology↗

bioRxiv2022

Opposite roles of Rad5 in DNA damage tolerance: playing in both error-free and mutagenic lesion bypass

DNA Damage Tolerance (DDT) functions to bypass replication-blocking lesions and is divided into two distinct pathways: error-prone Translesion Synthesis (TLS) and error-free Damage Avoidance (DA). Rad5 is a a multifunctional protein that is involved in these DDT processes. Rad5 contains three well defined domains: a RING domain that promotes PCNA polyubiquitination, a ssDNA-dependent ATPase/helicase domain, and a Rev1-binding domain. Both the RING domain and the ATPase/helicase domain are conserved in human Rad5 ortholog HLTF. In this study we used domain-specific mutants to address the contribution of each of Saccharomyces cerevisiae Rad5 functions to lesion tolerance. Using an assay based on the insertion of a single lesion into a defined locus in the genome of a living yeast cell, we demonstrate that Rad5 plays two opposite roles in lesion tolerance: i) Rad5 favors error-free lesion bypass by activating template switching through polyubiquitination of PCNA; ii) Rad5 is required for TLS by recruiting the TLS polymerase Rev1. We also show that the helicase activity does not play any role in lesion tolerance.

molecular biology↗

bioRxiv2021

Extending the gap and loading RecA: the presynaptic phase plays a pivotal role in modulating lesion tolerance pathways

During replication, the presence of unrepaired lesions results in the formation of single stranded DNA (ssDNA) gaps that need to be repaired to preserve genome integrity and cell survival. All organisms have evolved two major lesion tolerance pathways to continue replication: Translesion Synthesis (TLS), potentially mutagenic, and Homology Directed Gap Repair (HDGR), that relies on homologous recombination. In Escherichia coli, the RecF pathway repairs such ssDNA gaps by processing them to produce a recombinogenic RecA nucleofilament during the presynaptic phase. In this study, we show that the presynaptic phase is crucial for modulating lesion tolerance pathways. Indeed, impairing either the extension of the ssDNA gap (mediated by the nuclease RecJ and the helicase RecQ) or the loading of RecA (mediated by the RecFOR complex) leads to a decrease in HDGR. We suggest a model where defects in the presynaptic phase delay the formation of the D-loop and increase the time window allowed for TLS. We indeed observe an increase in TLS independent of SOS induction. In addition, we revealed an unexpected synergistic interaction between recF and recJ genes, that results in a recA deficient-like phenotype in which HDGR is almost completely abolished.

molecular biology↗

bioRxiv2021

Eukaryotic stress induced mutagenesis is limited by a local control of Translesion Synthesis

The DNA Damage Response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this report, we raise the question whether TLS is controlled locally, or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that: i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, ii) a single lesion is enough to induce PCNA ubiquitination, and iii) PCNA ubiquitination is an imperative requirement for TLS to occur. More importantly, we show that global PCNA ubiquitination that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis.

molecular biology↗

bioRxiv2020

Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis

DNA replication is mediated by the coordinated actions of multiple enzymes within replisomes. Processivity clamps tether many of these enzymes to DNA, allowing access to the primer/template junction. Many clamp-interacting proteins (CLIPs) are involved in genome maintenance pathways including translesion synthesis (TLS). Despite their abundance, DNA replication in bacteria is not perturbed by these CLIPs. Here we show that while the TLS polymerase Pol IV is largely excluded from moving replisomes, the remodeling of ssDNA binding protein (SSB) upon replisome stalling enriches Pol IV at replication forks. This enrichment is indispensable for Pol IV-mediated TLS on both the leading and lagging strands as it enables Pol IV-processivity clamp binding by overcoming the gatekeeping role of the Pol III epsilon subunit. As we have demonstrated for the Pol IV-SSB interaction, we propose that the binding of CLIPs to the processivity clamp must be preceded by interactions with factors that serve as localization markers for their site of action.

biochemistry↗

bioRxiv2019

iDamage: a method to integrate modified DNA into the yeast genome

In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the yeast genome, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of rad51 recombinase) or through its control by preventing the poly-ubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of TLS. Such regulatory aspects of the DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will allow exploration of many biological processes related to replication and repair of modified DNA.

molecular biology↗

bioRxiv2017

DNA lesions proximity modulates damage tolerance pathways

The genome of all organisms is constantly threatened by numerous agents that cause DNA damages. When the replication fork encounters an unrepaired DNA lesion, two DNA damage tolerance pathways are possible: error-prone translesion synthesis (TLS) that requires specialized DNA polymerases, and error-free Damage Avoidance (DA) that relies on homologous recombination. The balance between these two mechanisms is essential since it defines the level of mutagenesis during lesion bypass, allowing genetic variability and adaptation to the environment, but also introducing the risk of generating genome instability. Here we report that the mere proximity of replication-blocking lesions that arise in Escherichia colis genome during a genotoxic stress, leads to a strong increase in the use of the error-prone TLS. We show that this increase is caused by the local inhibition of homologous recombination due to the overlapping of single-stranded DNA regions generated downstream the lesions. This increase in TLS is independent of SOS activation, but its mutagenic effect is additive with the one of SOS. Hence, the combination of SOS induction and lesions proximity leads to a strong increase in TLS that becomes the main lesion tolerance pathway used by the cell during a genotoxic stress.

molecular biology↗

bioRxiv2017

A non-catalytic role of RecBCD in Homology Directed Gap Repair and Translesion Synthesis

The RecBCD complex is a key factor in DNA metabolism. This protein complex harbors a processive nuclease and two helicases activities that give it the ability to process duplex DNA ends. These enzymatic activities make RecBCD a major player in double strand break repair, conjugational recombination and degradation of linear DNA. In this work, we unravel a new role of the RecBCD complex in the processing of DNA single-strand gaps that are generated at DNA replication-blocking lesions. We show that independently of its nuclease or helicase activities, the entire RecBCD complex is required for recombinational repair of the gap and efficient translesion synthesis. Since none of the catalytic functions of RecBCD are required for those processes, we surmise that the complex acts as a structural element that stabilizes the blocked replication fork, allowing efficient DNA damage tolerance.

microbiology↗