Abstract
DNA replication errors are a major source of genome instability in all organisms. In the fission yeast Schizosaccharomyces pombe, the DNA damage response protein Brc1 binds phospho-histone H2A (γH2A)-marked chromatin during S-phase, but how Brc1 protects genome integrity remains unclear. Here we report that the non-homologous end-joining (NHEJ) protein Ku becomes critical for survival of replication stress in brc1∆ cells. Ku’s protective activity in brc1∆ cells does not involve its canonical NHEJ function or its roles in protecting telomeres or shielding DNA ends from Exo1 exonuclease. In brc1∆ pku80∆ cells, nuclear foci of Rad52 homologous recombination (HR) protein increase and Mus81-Eme1 Holliday junction resolvase becomes critical, indicating increased replication fork instability. Ku’s localization at a ribosomal DNA replication fork barrier associated with frequent replisome-transcriptosome collisions increases in brc1∆ cells and increased collisions correlate with an enhanced requirement for Brc1. These data indicate that Ku stabilizes replication forks in the absence of Brc1.
Highlights
Genome integrity is especially vulnerable during the DNA synthesis (S) phase of the cell cycle, when replisomes encounter DNA lesions, DNA-bound proteins and opposing transcriptosomes
We investigated whether Ku co-localizes with stalled forks. For these studies we focused on the replication fork barriers (RFBs) in the ribosomal DNA loci because we had previously found that the majority of spontaneous Brc1 foci co-localize with the nucleolus, which contains the rDNA occurring as tandem repeats in the subtelomeric arms of chromosome 3 [8, 43, 44]
This genetic interaction suggested that brc1Δ cells suffer increased double-strand breaks (DSBs) that require non-homologous end-joining (NHEJ) for repair, but we found that Ligase IV was not required in brc1Δ cells
Summary
Genome integrity is especially vulnerable during the DNA synthesis (S) phase of the cell cycle, when replisomes encounter DNA lesions, DNA-bound proteins and opposing transcriptosomes. Genome maintenance proteins insure accurate genome duplication during replicative stress, with the paramount factor being the master checkpoint protein kinase known as ATR in humans, Mec in the budding yeast Saccharomyces cerevisiae and Rad in the fission yeast Schizosaccharomyces pombe [1,2,3,4]. The carboxyl terminus of histone H2A in yeasts and H2AX in mammals is a key substrate of these checkpoint kinases [5]. PhosphoH2A/X (γH2A/X) is best known for its functions at double-strand breaks (DSBs) but it marks a diverse array of genomic features during S-phase in fission yeast, including natural replication fork barriers, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats [6]. A key role of γH2A in S-phase was revealed by the discovery that Brc genome protection protein forms nuclear foci by binding γH2A during
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