Genome-wide mapping of histone H2A phosphorylation during S-phase in Schizosaccharomyces pombe (fission yeast)

Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (gH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for DNA repair/checkpoint proteins. Our data showed that gH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. The enrichment of gH2A at these sites was confirmed by multiple ChiP-qPCR experiments. Phosphorylated Histone H2A was ChIPed with anti-gammaH2A antibody vs. Input(whole cell extract) in S. pombe cells synchronized in S-phase.