The striking sensitivity of H2A.Z mutants to DSB-inducing agents (Fig. 5) implied that H2A.Z is indeed functionally linked to DSB repair. To assess H2A.Z’s exact role in DSB repair, a donor-proficient strain was used in which a DSB is only shortly induced at MAT and subsequently repaired by gene conversion. In this strain, repair involves the homologous HMLα locus and results in mating type switching from MATa to MATα. Therefore, the kinetics of repair can be readily followed by PCR or Southern blot with MATα-specific primers or probes, respectively. Indeed, when quantified by RT-PCR, mutants lacking H2A.Z showed a significant delay in mating type switching by gene conversion (Fig. 9A and 9B). However, Southern blot analysis demonstrated that H2A.Z, apart from slightly influencing the kinetics, is definitely proficient in mating type switching per se (Fig. 9B and 9C). The switched
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Results Role of H2A.Z in DSB repair
Figure 9. Effect of H2A.Z deletion on DSB repair by gene conversion
(A) RT-PCR with primer pair PA and PB as depicted in (B). PCR product formation indicates strand-
invasion into the homologous region at HML and mating type switching. Obtained signal intensities were normalized to an unaffected control locus and the WT 4h time point was set to 100%. Data are shown as mean of 3 independent experiments ±SEM.
(B) Schematic representation of Chromosome III before (top panel) and after (lower panel) mating type switching in the donor proficient strain used to monitor DSB repair by gene conversion in (A) and (C). A single DSB (arrow) is induced at the MAT-locus by expression of HO endonuclease. Regions of homology and the recombination enhancer (RE) element are shown as boxes. Position of primer pair PA and PB used for assaying mating type switching in (B) are indicated (blue). StyI restriction sites, the
resulting DNA fragments and the probe used for Southern blot detection in (C) are indicated.
(C) Southern blot analysis of mating type switching. Genomic DNA prepared from samples taken at the indicated time points was digested with StyI and run on an alkaline gel followed by gel blotting and hybridization with the MAT-specific probe indicated in (B). HO endonuclease cuts the intact MATa fragment (purple, 730 bp) giving rise to a smaller cleavage fragment (red, 508 bp). The switched, MATα
product lacks the StyI restriction site present within the MATa sequence and is thus larger (green, 1881 bp). The loading control corresponds to the first StyI restriction fragment on the right of the MAT locus (yellow, 4326 bp), which is not affected by mating type switching.
product restriction fragment appeared in both Δhtz1 and Δswr1. Of note, yeast
mating type switching requires only about 300 bp of DNA to the right of the HO cut site (containing the homology information) to be resected in order for repair to ensue. Thus e.g. mutations in the MRX complex which is required for resection, only delay but do not prevent mating type switching (Ivanov et al., 1994). A stricter requirement for resection can be seen in the single-strand annealing (SSA) pathway, where an induced DSB is repaired by recombination between flanking repeats (Fig. 10A and Vaze et al., 2002). In this assay, galactose-inducible HO expression creates
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Results Role of H2A.Z in DSB repair
Figure 10. Role of H2A.Z in DSB repair by single strand annealing (SSA)
(A) Schematic representation of DSB repair by SSA in the tester strain YMV45. An HO cut site was introduced into the endogenous leu2 locus and a second LEU2 gene copy placed 4.6kb upstream. A single DSB (arrow) is induced at the leu2-locus by galactose-induced expression of HO endonuclease. 5’-strand resection needs to proceed for at least 4.6 kb for single strand annealing between the complementary regions to occur. The annealed intermediate is then processed by removal of the overhanging single-stranded tails, gap filling and ligation.
(B) Equal amounts of cells were spotted onto plates containing either galactose (YPGal) or glucose (control) as carbon source. Images were taken after 48h of growth at 30˚C.
a DSB within the endogenous leu2 gene locus. As the second LEU2 sequence is inserted 4.6 kb downstream, resection of this stretch of DNA is a prerequisite for SSA to occur. To test whether mutants lacking H2A.Z were proficient in SSA, WT and mutant tester strains were spotted onto galactose-containing plates (Fig. 10B). Successful completion of SSA between repeats eliminates the HO cut site, and cells can therefore grow on galactose-containing plates. However, strains incapable of repair by SSA have to continually cope with a persistent DSB and eventually die due to chromosome loss. Similar to known resection mutants like Δmre11 and Δsgs1
(Vaze et al., 2002), Δhtz1 cells are indeed extremely sensitive to HO-expression
when productive SSA is required for survival (Fig. 10B). Importantly, Δhtz1, which is
known to play a role in GAL-gene transcription, does not confer galactose sensitivity per se as shown by the robust growth in a DF5 WT background (Fig 6B, right panel). Recent reports have revealed at least two additional pathways that are required for proper resection in the absence of, or in addition to the MRX-complex (Gravel et al., 2008; Mimitou and Symington, 2008; Zhu et al., 2008). While MRX is responsible for the initiation of resection, long-range resection is facilitated in an Sgs1- and Exo1-dependent manner. Interestingly, the effect of Δhtz1 on SSA was
Results Role of H2A.Z in DSB repair additive when combined with mutations in Mre11 and Exo1. However, the double mutant Δhtz1Δsgs1 showed the same sensitivity as, i.e. was epistatic to, Δhtz1
alone. This suggests that H2A.Z may promote resection in a pathway together with Sgs1 and in parallel to MRX and Exo1.