Abstract
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 (γH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, γH2A 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. γH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that γH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to γH2A 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.
Highlights
During DNA replication cells are vulnerable to loss of genetic information and mutation [1]
Since cH2A ChIP-on-chip analysis has not been performed in S. pombe, we first tested if cH2A could be detected at a site-specific DNA doublestrand breaks (DSBs) made by activating expression of the HO-endonuclease [32]. cH2A ChIP was performed with a phospho-H2A specific antibody [33]
Mat1 is replicated from the right side, which induces a DSB at a fragile site next to mat1 [35,36,37]
Summary
During DNA replication cells are vulnerable to loss of genetic information and mutation [1]. The DNA replication checkpoint pathway monitors the genome to detect and stabilize stalled forks, initiate repair, and delay mitotic entry until DNA damage is repaired [2]. Checkpoint activation during replication is triggered by the kinase ATR, which is crucial for maintenance of genome stability during S-phase [3]. Candidates include highly repetitive DNA, natural replication fork barriers (RFBs), and chromosomal ‘‘fragile sites’’, which may present obstacles to replication [1,4,5]. It is unknown whether specific chromosomal domains are responsible for Rad activation during an unperturbed cell cycle
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