Microarray-based genetic screen defines SAW1, a gene required for Rad1/Rad10-dependent processing of recombination intermediates

Abstract

Elimination of a double-strand break (DSB) flanked by direct repeat sequences is mediated by single-strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair, and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA with a plasmid-based SSA assay coupled to a barcode microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins, and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3' flap cleavage step. Slx4 and Saw1 also contribute to the integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2, are defective in 3' flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.

Figure 1. Slx4 and Saw1 are needed for efficient repair of a DSB flanked by direct repeat sequences on a chromosome

A. SSA between 205 bp ura3 repeats at yeast chromosome V. Survival rate after HO endonuclease induced DSB (C) and Southern blot hybridization of BglII digested genomic DNA (E) with radio-labeled probe next to the URA3 gene to detect SSA product formation are shown for wild type tNS1379 strain and its mutant derivatives. B. SSA between 1.3 kb leu2 repeats that located 24 kb apart on chromosome III. Survival rate of wild type and the SLX4, SAW1, or RAD1 gene deletion derivatives upon HO induction (D) were shown. Data represent means ± standard deviation (s.d.) from three independent experiments.

Figure 2. DNA damage sensitivity profiles of SSA mutants

Serial dilutions of each strain were plated onto YEPD with or without indicated concentration of MMS, HU, and phleomycin. To measure UV sensitivity, strains were irradiated immediately after plating onto YEPD using a Stratalinker UV source. Plates were incubated for 3 to 4 days at 30 °C.

Figure 3. Deletion of SLX4 or SAW1 reduced integrity of ribosomal DNA (rDNA) arrays

A. rDNA recombination assay. A circle is a centromere of chromosome XII. Gray boxes are telomeres. B. Wild type and SIR2 gene deletion derivatives carrying an ADE2 marker integrated into the rDNA array were grown overnight and then plated onto YEPD plate. Colonies were allowed to grow for 48 h at 30°C then placed at 4°C for additional 48 h. The number of half-red half-white colonies that represent a marker loss event during the first cell division after plating were divided by the total number of colonies to calculate rDNA recombination rate. The t-test was used for statistical analysis.

Figure 4. Kinetics of 3’ non-homologous tail appearance and removal in SSA

A. qPCR-based 3’-flap detection assay. Direct repeats (grey) and primers used for qPCR (arrows) are shown. B. Kinetics of 3’-flaps in tNS1379 (wild type) and various mutants. Genomic DNA from various mutants was digested by Sau3AI, and used for qPCR with primer sets that anneal to a 3’-flap. PCR results at multiple time intervals after HO expression using a primer set that anneal to a 3’-flap were normalized by control PCR with primers that anneal to ACT1 or PRE1, were shown to indicate kinetics of 3’-flap formation and removal. Data represent means ± s.d. from at least three independent experiments.

Figure 5. Physical interaction among SSA proteins

A. Yeast two-hybrid assays were performed with GAL4 DNA binding domain (GBD) fusion of Slx4 and activation domain (GAD) fusion of Rad1 to detect interaction between these proteins. B. Pull-down assay with GST-Saw1. Yeast crude extract expressing TAP tagged proteins were incubated with GST-Saw1 coated beads for 2 h. After extensive washing with 20 volume of PBS + 0.2% Triton X-100, beads were treated with protein loading buffer and loaded on SDS-PAGE. TAP tagged proteins were detected by PAP reagent (Sigma). C. Saw1 interacts with Rad1 in vivo. FLAG Tagged Saw1 or Rad1 protein was pull down by anti FLAG or anti-Rad1 antibody and the immunoprecipitates were probed with anti-Rad1 or anti-FLAG antibody. D. Saw1 interacts with Msh2 and Rad52. Yeast strain expressing Saw1-TAP and Rad52-13Myc was treated first with 0.1 % MMS for 0.5 h and immunoprecipitated with IgG agarose. The pull-down proteins were hybridized with anti-Rad1, anti-Msh2 or anti-Myc antibodies. E, F, and G. ³⁵S-labeled Rad1, C-terminal nuclease domain deleted rad1 mutant proteins (rad1ΔC), Msh2, and Rad52 obtained by coupled in vitro transcription-translation, were incubated with GST-Saw1 or GST. Proteins associated with GST-Saw1 or GST were isolated on glutathione-Sepharose beads, resolved by SDS-PAGE, and visualized by autoradiography.

Figure 6. Interaction between Rad1 and Saw1 is critical for 3’-flap removal in recombination

A. Positions of deletions in saw1 mutants. B. The effect of saw1 mutations on 3’-flap removal was tested using plasmid based flap removal assay with pFP120 carrying 308 and 610 bp of flaps. The percent plasmid retention were plotted for SAW1 deletion carrying wild type SAW1, saw1Δ_18–24 or saw1Δ_159–169 mutations. Data represent means ± s.d. from at least three independent experiments. C. Pull-down assay was performed to detect interaction between GST-Saw1 and GST-Saw1 mutants with Rad1 Msh2-TAP, Rad52-13Myc proteins. D. The effect of saw1 mutations on SSA between 205 bp ura3 direct repeat sequences flanked by a HO endonuclease-induced DSB. Southern blot hybridization of BglII digested genomic DNA with the radiolabeled probe next to the URA3 gene was performed to detect SSA product formation in SAW1 gene deletion strain expressing saw1 mutants from yeast centromeric plasmid.

Figure 7. Recruitment of Rad1 at the recombination intermediate carrying 3’-flaps requires Saw1 proteins

A. HO cleavage and the location of primers (arrows) used in the ChIP assay to detect Rad1 and the nuclease deficient rad1 derivative (rad1-D825A) at the recombination intermediate carrying 3’-flap. The levels of Rad1-3HA or rad1-D825A-3HA in wild type or saw1Δ at the DSB were detected by ChIP assay using an anti-HA antibody. Chromatin was isolated at the indicated time after galactose addition, crosslinked, and fragmented by sonication. After immunoprecipitation and reverse crosslinking, purified DNA was analyzed by qPCR using four sets of primers that anneal 0.2 kb (B, JC1/JC2), 2.4 kb (C, JC3/JC4), and 0.5 kb (D, JC5/JC6) to the DSB, as well as primers specific for the PRE1 gene situated on chromosome V as a control. PCR signals from each primer set at different durations of HO expression were quantified and plotted as a graph. IP represents the ratio of the Rad1 PCR signal before and after HO induction, normalized by the PCR signal of the PRE1 control. Each point is the average of two separate experiments.