Dynamics of Yeast Histone H2A and H2B Phosphorylation in Response to a Double‐Strand Break

Abstract

In Saccharomyces cerevisiae, a single double‐strand break (DSB) triggers extensive phosphorylation of histone H2A (known as γ‐H2AX) over ≥ 50 kb on either side of the DSB. This modification is carried out by either Tel1, the ATM homolog, or Mec1, the ATR homolog. We have recently described a second DSB‐induced modification, γ‐H2B, the phosphorylation of the C terminal T129 residue of histone H2B. To understand in detail how γ‐H2AX and γ‐H2B spread along the chromosome from a DSB, we have utilized chromatin immunoprecipitations followed by tiling arrays. γ‐H2AX and γ‐H2B modifications are similar, but there is a marked absence of γ‐H2B near telomeres. We find that there is reduced γ‐H2AX and γ‐H2B modification over strongly transcribed regions and the spreading of γ‐H2AX at the region behind is also impaired. When transcription of the galactose‐regulated genes is turned off by the addition of glucose, γ‐H2AX is restored within 5 min; when these genes are again induced, γ‐H2AX is rapidly lost. The restoration of γ‐H2AX in transcribed regions can be done by Tel1 alone even 7 h after break induction, suggesting that Tel1 remains associated with damaged chromosomes for an extended time. In addition, we show that if a DSB is generated 14 kb from CEN2, γ‐H2AX takes place at regions around all the other centromeres. This modification in trans can be observed even in the absence of formaldehyde crosslinking of the samples.