As yeast are starved of nutrients, they enter G0, a quiescent state. Quiescent yeast (Q) cells retain viability for extended periods of time and resume growth following supplementation of missing nutrients. As such, Q cells have become a valuable model for studying longevity and self-renewal of chronologically aged cells. Traditional isolation of Q cells involves a relatively long centrifugation time through a continuous density gradient. Here, we describe a rapid and cost-effective Q-cell isolation technique that uses a single-density, one-step gradient prepared from media containing iodixanol.

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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.

Nearly 50 hereditary diseases result from the inheritance of abnormally long repetitive DNA microsatellites. While it was originally believed that the size of inherited repeats is the key factor in disease development, it has become clear that somatic instability of these repeats throughout an individual’s lifetime strongly contributes to disease onset and progression. Importantly, somatic instability is commonly observed in terminally differentiated, postmitotic cells, such as neurons. To unravel the mechanisms of repeat instability in nondividing cells, we created an experimental system to analyze the mutability of Friedreich’s ataxia (GAA)n repeats during chronological aging of quiescent Saccharomyces cerevisiae. Unexpectedly, we found that the predominant repeat-mediated mutation in nondividing cells is large-scale deletions encompassing parts, or the entirety, of the repeat and adjacent regions. These deletions are caused by breakage at the repeat mediated by mismatch repair (MMR) complexes MutSβ and MutLα and DNA endonuclease Rad1, followed by end-resection by Exo1 and repair of the resulting double-strand breaks (DSBs) via nonhomologous end joining. We also observed repeat-mediated gene conversions as a result of DSB repair via ectopic homologous recombination during chronological aging. Repeat expansions accrue during chronological aging as well—particularly in the absence of MMR-induced DSBs. These expansions depend on the processivity of DNA polymerase δ while being counteracted by Exo1 and MutSβ, implicating nick repair. Altogether, these findings show that the mechanisms and types of (GAA)n repeat instability differ dramatically between dividing and nondividing cells, suggesting that distinct repeat-mediated mutations in terminally differentiated somatic cells might influence Friedreich’s ataxia pathogenesis.

DNA are sites of genomic instability. Long CAG/CTG repeats form hairpin structures, are fragile, and can expand during DNA repair. The chromatin response to DNA damage can influence repair fidelity, but the knowledge of chromatin modifications involved in maintaining repair fidelity within repetitive DNA is limited. In a screen for CAG repeat fragility in Saccharomyces cerevisiae, histone 2A copy 1 (H2A.1) was identified to protect the repeat from increased rates of breakage. To address the role of H2A in CAG repeat instability, we tested the effect of deleting each histone H2 subytpe. Whereas deletion of HTA2, HTZ1, HTB1, and HTB2 did not significantly affect CAG repeat maintenance, deletion of HTA1 resulted in increased expansion frequency. Notably, mutation of threonine 126, unique to H2A.1, to a non-phosphorylatable alanine increased CAG repeat instability to a similar level as the hta1Δ mutant. CAG instability in the absence of HTA1 or mutation to hta1-T126A was dependent on the presence of the homologous recombination (HR) repair proteins Rad51, Rad52, and Rad57, and the Polδ subunit Pol32. In addition, sister chromatid recombination (SCR) was suppressed in the hta1Δ and hta1-T126A mutants and this suppression was epistatic to pol32Δ. Finally, break-induced replication (BIR) is impaired in the hta1Δ mutant, resulting in an altered repair profile. These data reveal differential roles for the H2A subtypes in DNA repair and implicate a new role for H2A.1 threonine-126 phosphorylation in mediating fidelity during HR repair and promoting SCR. Using a fragile, repetive DNA element to model endogenous DNA damage, our results demonstrate that H2A.1 plays a greater role than H2A.2 in promoting homology-dependent repair, suggesting H2A.1 is the true homolog of mammalian H2AX, whereas H2A.2 is functionally equivalent to mammalian H2A.Author Summary CAG/CTG trinuncleotide repeats are fragile sequences that when expanded can cause human disease. To evaluate the role of S. cerevisiae histone H2A copies in DNA repair, we have measured instability of an expanded CAG/CTG repeat tract and repair outcomes in H2A mutants. Although the two copies of H2A are nearly identical in amino acid sequence, we found that the CAG repeat is more unstable in the absence of H2A copy 1 (H2A.1) than H2A copy 2, and that this role appears to be partially dependent on a phosphorylatable threonine at residue 126 in the C-terminal tail of H2A.1. Further, we show through a series of genetic assays that H2A.1 plays a role in promoting homologous recombination events, including sister chromatid recombination and break-induced replication. Our results uncover a role for H2A.1 in mediating fidelity of repair within repetitive DNA, and demonstrate that modification of its unique Thr126 residue plays a role in regulating SCR. Given the dependence of HR repair on H2A.1 but not H2A.2, we conclude that H2A.1 plays a greater repair-specific role in the cell and therefore would be the true homolog of mammalian H2AX.