Rad3ATR Decorates Critical Chromosomal Domains with gH2A to Protect Genome Integrity during S-Phase in Fission Yeast

• Published in: PLoS genetics 2010-07, Vol.6 (7)
• Main Authors: Rozenzhak, Sophie, Mejia-Ramirez, Eva, Williams, Jessica S, Schaffer, Lana, Hammond, Jennifer A, Head, Steven R, Russell, Paul
• Format: Article
• Language: English
• Subjects: Schizosaccharomyces pombe
• ISSN: 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1001032

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 Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, 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. gH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that gH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to gH2A is crucial in the absence of Rqh1Sgs1, a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund-Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity. Eukaryotic genomes, which range in size from 6107 to 61011 base pairs, are replicated with nearly absolute fidelity every cell cycle. This amazing feat happens despite the frequent stalling or collapse of replication forks. The checkpoint kinase ATR is activated by replication fork stalling and phosphorylates histone H2A in nucleosomes surrounding damaged DNA. As the genomic regions triggering ATR activation are largely unknown, we used a whole-genome microarray to map chromosomal domains enriched with phospho-H2A during DNA replication in fission yeast. This analysis identified specific sites, including natural replication fork barriers in ribosomal DNA repeats, retrotransposon elements, and most surprisingly, all heterochromatin regions. Phospho-H2A binds the genome maintenance protein Brc1, and our genetic studies reveal that this molecular pathway becomes crucial in the absence of Rqh1, a conserved DNA helicase that is l