Abstract
CAG/CTG trinuncleotide repeats are fragile sequences that when expanded form DNA secondary structures and cause human disease. We evaluated CAG/CTG repeat stability and repair outcomes in histone H2 mutants in S. cerevisiae. Although the two copies of H2A are nearly identical in amino acid sequence, CAG repeat stability depends on H2A copy 1 (H2A.1) but not copy 2 (H2A.2). H2A.1 promotes high-fidelity homologous recombination, sister chromatid recombination (SCR), and break-induced replication whereas H2A.2 does not share these functions. Both decreased SCR and the increase in CAG expansions were due to the unique Thr126 residue in H2A.1 and hta1Δ or hta1-T126A mutants were epistatic to deletion of the Polδ subunit Pol32, suggesting a role for H2A.1 in D-loop extension. We conclude that H2A.1 plays a greater repair-specific role compared to H2A.2 and may be a first step towards evolution of a repair-specific function for H2AX compared to H2A in mammalian cells.
Highlights
Integral to the eukaryotic DNA damage response is chromatin structure modifications surrounding the break site
To identify important factors for maintaining expanded containing a YAC with a (CAG)/CTG trinucleotide repeats, a screen was performed for genes that protect against repeat fragility using a yeast artificial chromosome (YAC) end loss assay in the Mata haploid deletion set
Stemming from an initial observation in a genetic screen that CAG repeat fragility and instability were elevated in an hta1D mutant but not in an hta2D mutant, we demonstrated that H2A.1 and H2A.2 differentially contribute to homology-mediated repair
Summary
Integral to the eukaryotic DNA damage response is chromatin structure modifications surrounding the break site (reviewed in House et al, 2014a; Seeber et al, 2013; Price and D’Andrea, 2013). In response to DNA double strand breaks (DSBs) and stalled or collapsed replication forks, the SQEL motif in the H2A C-terminal tail is phosphorylated at Ser129 (H2AX-Ser139 in mammals) by the Phosphoinositide 3-Kinase-Related Kinases (PIKKs), Mec and Tel (ATR and ATM in mammals) (Downs et al, 2000; Lisby et al, 2004) This modification, termed gH2A (gH2AX in mammals), marks the site of damage and is propagated along the chromatin, detectable up to 50 kb from the break site in yeast (Shroff et al, 2004) and megabases in mammalian cells (Rogakou et al, 1999).
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