Rad3ATR Decorates Critical Chromosomal Domains with γH2A to Protect Genome Integrity during S-Phase in 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 (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

Establishment of Cohesion at the Pericentromere by the Ctf19 Kinetochore Subcomplex and the Replication Fork-Associated Factor, Csm3

The cohesin complex holds sister chromatids together from the time of their duplication in S phase until their separation during mitosis. Although cohesin is found along the length of chromosomes, it is most abundant at the centromere and surrounding region, the pericentromere. We show here that the budding yeast Ctf19 kinetochore subcomplex and the replication fork-associated factor, Csm3, are both important mediators of pericentromeric cohesion, but they act through distinct mechanisms. We show that components of the Ctf19 complex direct the increased association of cohesin with the pericentromere. In contrast, Csm3 is dispensable for cohesin enrichment in the pericentromere but is essential in ensuring its functionality in holding sister centromeres together. Consistently, cells lacking Csm3 show additive cohesion defects in combination with mutants in the Ctf19 complex. Furthermore, delaying DNA replication rescues the cohesion defect observed in cells lacking Ctf19 complex components, but not Csm3. We propose that the Ctf19 complex ensures additional loading of cohesin at centromeres prior to passage of the replication fork, thereby ensuring its incorporation into functional linkages through a process requiring Csm3

Topoisomerase I poisoning results in PARP-mediated replication fork reversal

Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics