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Local regulation of the Srs2 helicase by the SUMO-like domain protein Esc2 promotes recombination at sites of stalled replication

ABSTRACT: Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage-bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage-bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domains-containing Saccharomyces cerevisiae protein, Esc2, facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause sub-optimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress, while globally prevented at undamaged replicating chromosomes.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE65701 | GEO | 2015/02/16

SECONDARY ACCESSION(S): PRJNA274743

REPOSITORIES: GEO

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Multifaceted down-regulation of the Srs2 helicase by the SUMO-like domain protein Esc2 promotes local recombination at sites of stalled replication

Project description:The SUMO-like domains-containing family of proteins to which Saccharomyces cerevisiae Esc2 belongs facilitates DNA damage tolerance (DDT) via elusive mechanisms. Here we report that Esc2 promotes recombination-mediated DDT by engaging in functional and physical interactions with Srs2 and Elg1, two readers of SUMOylated PCNA and modulators of DDT. These interactions depend on the SUMO Interacting Motifs of Elg1 and Srs2, and on the SUMO-like domains of Esc2. Mechanistically, Esc2 promotes Elg1 association to damaged and stalled forks and Srs2 turnover. Elg1 limits the levels of SUMOylated PCNA and subsequently of the anti-recombinase Srs2 at damaged sites, upholding local Rad51 binding. In conjunction with the SUMO–targeted ubiquitin ligase Slx5/Slx8, Esc2 also promotes proteasome-mediated Srs2 turnover, a process further enhanced by CDK-mediated Srs2 phosphorylation. Our results provide mechanistic insights into how SUMO- and DNA damage response-regulated pathways intersect to enable local error-free damage-bypass by recombination in the face of genotoxic stress. Proteins ChIP-chip analyses analysis were carried out as described (Bermejo et al., 2009). Labelled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.

2015-02-16 | E-GEOD-65701 | biostudies-arrayexpress

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The Mgs1/WRNIP1 ATPase is required to prevent a recombination salvage pathway at damaged replication forks

Project description:DNA damage tolerance (DDT) is crucial for genome integrity maintenance. DDT is mainly carried out by template switch recombination, an error-free mode of overcoming DNA lesions, or translesion DNA synthesis, which is error-prone. Here we investigated the role of Mgs1/WRNIP1 in modulating DDT. Using budding yeast, we found that elimination of Mgs1 in cells lacking Rad5, an essential protein for DDT, activates an alternative mode of DNA damage bypass, driven by recombination, that allows chromosome replication and cell viability under stress conditions that block DNA replication forks. This salvage pathway is RAD52- and RAD59-dependent, requires the DNA polymerase and PCNA-modification at K164, and is enabled by Esc2 and the PCNA-unloader Elg1, being inhibited when Mgs1 is present. We propose that Mgs1 is necessary to prevent a potentially toxic recombination salvage pathway at sites of perturbed replication, which in turn favors Rad5-dependent template switching, thus helping to preserve genome stability.

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PHOSPHORYLATION-DEPENDENT ASSOCIATION OF WRN WITH RPA IS REQUIRED FOR RECOVERY OF REPLICATION FORKS STALLED AT SECONDARY DNA STRUCTURES

Project description:The WRN protein mutated in the premature aging disorder Werner syndrome is vital for metabolism of perturbed replication forks. Replication Protein A (RPA) robustly enhances WRN helicase activity in specific cases when tested in vitro. However, the significance of RPA-binding to WRN in vivo has remained largely unexplored. We identified several conserved phosphorylation sites in the acidic domain of WRN targeted by Casein Kinase 2 (CK2). These sites are crucial for WRN-RPA interaction in vitro and in human cells. Using the CK2-unphosphorylatable WRN mutant lacking the ability to bind RPA, we determined that the WRN-RPA complex plays a critical role in fork recovery after replication stress whereas is not necessary for the processing of replication forks or preventing DNA damage when forks stall or collapse. RPA-binding by WRN and its helicase activity are crucial for countering the persistence of G4 structures after fork stalling. Absence of WRN-RPA binding hampers fork recovery, causing single-strand DNA gaps, enlarged by MRE11, and triggering MUS81-dependent double-strand breaks which requires efficient repair by RAD51 to prevent excessive DNA damage.

| MSV000095749 | MassIVE

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