Deposition of histone H2A.Z by the SWR-C remodeling enzyme prevents genome instability

Abstract

The yeast SWR-C chromatin remodeling enzyme catalyzes chromatin incorporation of the histone variant H2A.Z which plays roles in transcription, DNA repair, and chromosome segregation. Dynamic incorporation of H2A.Z by SWR-C also enhances the ability of exonuclease I (Exo1) to process DNA ends during repair of double strand breaks. Given that Exo1 also participates in DNA replication and mismatch repair, here we test whether SWR-C influences DNA replication fidelity. We find that inactivation of SWR-C elevates the spontaneous mutation rate of a strain encoding a L612M variant of DNA polymerase (Pol) δ, with a single base mutation signature characteristic of lagging strand replication errors. However, this genomic instability does not solely result from reduced Exo1 function, because single base mutator effects are seen in both Exo1-proficient and Exo1-deficient pol3-L612M swr1Δ strains. The data are consistent with the possibility that incorporation of the H2A.Z variant by SWR-C may stimulate Exo1 activity, as well as enhance the fidelity of replication by Pol δ, the repair of mismatches generated by Pol δ, or both.

Keywords: Chromatin remodeling; Exonuclease 1; Genome instability; H2A.Z; Mismatch repair.

Fig. 1

H2A.Z deposition by SWR-C reduces the mutation rate due to pol3-L612M. Spontaneous mutation rates to 5-FOA resistance (A) and canavanine resistance (B) with 95% confidence intervals are shown. Statistically significant differences (p<0.0001) in mutation rate are indicated by an asterisk. Deletion of SWR1 or HTZ1 separately and in combination increases URA3 and CAN1 mutation rate in a pol3-L612M mutator background. (C) SWR1 was deleted in the pol3-L612M mutator strain in an exo1Δ background. Mutation rates at the URA3 (left panel) and CAN1 loci (right panel) are shown as in (A). (D) The effect of SWR1 deletion on URA3 mutation rate in wild type (WT) and polymerase mutator strains was calculated in an msh2Δ background. Mismatch repair is required for the mutagenic effect of SWR1 deletion.

Fig. 2

URA3 mutation spectra in pol3-L612M strains ± SWR1 and/or EXO1. The coding strand of the 804 base pair URA3 open reading frame (ORF) is shown with every tenth base indicated by a circle below the DNA sequence. Letters indicate single base substitutions, closed triangles indicate single base additions and open triangles indicate single base deletions. (A) Spectra for the pol3-L612M SWR1 and pol3-L612M swr1Δ strains are depicted above and below the URA3 ORF, respectively. (B) Spectra for the pol3-L612M SWR1 exo1Δ and pol3-L612M swr1Δ exo1Δ strains are depicted above and below the URA3 ORF, respectively.

Fig. 3

Mutation rates at four specific locations in URA3. (A) pol3-L612M ± SWR1 strains. Rates for the pol3-L612M SWR1 strain were calculated using data from [24] plus additional 5-FOA-resistant mutant sequences. No mutations were observed at URA3 positions 95 or 255 in this strain, so rates were calculated as ≤ values had one event been observed. Rates for the pol3-L612M swr1Δ mutant were calculated using data in Figure 2 and Table 1. (B) pol3-L612M exo1Δ ± SWR1 strains. Rates for the pol3-L612M SWR1 exo1Δ strain were calculated using data from [9] plus additional 5-FOA-resistant mutant sequences (this study). No mutations were observed at URA3 positions 345 or 768 in this strain, so rates were calculated as ≤ values had one event been observed. Rates for the pol3-L612M swr1Δ exo1Δ mutant were calculated using data in Figure 2 and Table 1.