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

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. Overall design: One INPUT (SUP) with the corresponding IP samples for each ChIP on chip. The following samples are processed. Elg1-FLAG, esc2Δ Elg1-FLAG, Rad51, esc2ΔRad51, Esc2-Myc, Esc2Δ154_198_Myc, Pol3-Myc, and Brdu. Cells were grown in 200 ml of YPD media at 25°C in 1L falsk for a density of 1.2×10^7 cells/ml. G1 synchronization was performed by α−factor treatment (2 μg/ml) for 150 minutes. Cells were pelleted and released in media containing 0.2 M HU at 25°C for 30 (Rad51), 60 min (Elg1-FLAG, Esc2-Myc, Esc2Δ154_198-Myc or 90 min (Pol3-Myc and Brdu) and fixed with 1% formaldehyde for 30 min or 120 (as indicated) min at room temperature. Proteins ChIP-chip analyses were carried out as described (Bermejo R, Katou YM, Shirahige K, Foiani M. ChIP-on-chip analysis of DNA topoisomerases. Methods Mol Biol. 2009;582:103-18). Labelled probes were hybridized to Affymetrix S. cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.