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

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. 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.