Figure 5.

Sae2-ms and Sae2-S134L enhance Mre11 and Tel1 association to DSBs and reduce hairpin cleavage. (A) ChIP analysis. HO was induced by galactose addition at time zero in exponentially growing JKM139 derivative cells. Relative fold enrichment of Mre11-Myc protein at the indicated distances from the HO cleavage site was determined after ChIP with anti-Myc antibodies and subsequent qPCR analysis. Plotted values are the mean values with error bars denoting SD (n = 3). * P < 0.05 (Student’s t-test). (B) As in (A), but showing relative fold enrichment of Tel1-HA after ChIP with anti-HA antibodies. (C and D) Western blot analysis with anti-Myc or anti-HA antibodies of protein extracts prepared from exponentially growing cells. The same amount of extracts was probed with anti-Pgk1 antibodies as loading control. (E) Recombination frequency of strains with the lys2-AluIR and lys2-Δ5’ ectopic recombination system. The rate of Lys⁺ recombinants was derived from the median recombination frequency. The reported values are the mean values with SD indicated in brackets (n = 3). (F) Exponentially growing cells were serially diluted (1:10) and spotted out onto YEPD plates with or without HU or MMS. All strains carried SML1 deletion that kept mec1∆ cells viable. (G) Protein extracts prepared from exponentially growing cells were analyzed by western blotting with anti-HA and anti-Myc antibodies either directly (Input) or after immunoprecipitation (IP) with anti-HA antibodies. *indicates a cross-hybridization signal.