Brc1-dependent recovery from replication stress

Abstract

Summary BRCT-containing protein 1 (Brc1) is a multi-BRCT (BRCA1 carboxyl terminus) domain protein in Schizosaccharomyces pombe that is required for resistance to chronic replicative stress, but whether this reflects a repair or replication defect is unknown and the subject of this study. We show that brc1D cells are significantly delayed in recovery from replication pausing, though this does not activate a DNA damage checkpoint. DNA repair and recombination protein Rad52 is a homologous recombination protein that loads the Rad51 recombinase at resected double-stranded DNA (dsDNA) breaks and is also recruited to stalled replication forks, where it may stabilize structures through its strand annealing activity. Rad52 is required for the viability of brc1D cells, and brc1D cells accumulate Rad52 foci late in S phase that are potentiated by replication stress. However, these foci contain the single-stranded DNA (ssDNA) binding protein RPA, but not Rad51 or cH2A. Further, these foci are not associated with increased recombination between repeated sequences, or increased post-replication repair. Thus, these Rad52 foci do not represent sites of recombination. Following the initiation of DNA replication, the induction of these foci by replication stress is suppressed by defects in origin recognition complex (ORC) function, which is accompanied by loss of viability and severe mitotic defects. This suggests that cells lacking Brc1 undergo an ORC-dependent rescue of replication stress, presumably through the firing of dormant origins, and this generates RPA-coated ssDNA and recruits Rad52. However, as Rad51 is not recruited, and the checkpoint effector kinase Chk1 is not activated, these structures must not contain the unprotected primer ends found at sites of DNA damage that are required for recombination and checkpoint activation. IntroductionDuring DNA replication, cells are extremely vulnerable to DNA damage. This vulnerability is due to both the genomic lesions themselves, but also to the impediment these lesions cause to the completion of replication. Replication forks that encounter sites of DNA damage stall in the face of these lesions, a process that activates the DNA replication checkpoint to help maintain stalled fork stability, halt the cell cycle, and initiate DNA repair. The majority of replication forks are presumed to retain the full complement of replication machinery in a stable conformation at sites of fork stalling until the impeding lesion is repaired. However, some lesions are not readily resolved and so alternative mechanisms for the completion of replication have evolved. These include the collapse of the stalled replication fork and its repair through homologous recombination (HR), bypass of the DNA lesion through the post-replication repair (PRR) pathway, which includes both error-prone and error-free mechanisms, and the completion of replication through the firing of adjacent origins (Branzei and Foiani, 2008;Branzei and Foiani, 2009).PRR mediates lesion bypass through a two-step process. Initia...

TORC2 Is Required to Maintain Genome Stability during S Phase in Fission Yeast

“…Similar results were obtained from studies of Brc1, a six-BRCT domain protein (52). ⌬brc1 cells show elevated levels of spontaneous Rad52-YFP foci (53) but reduced spontaneous recombination rates between direct repeats (52). Genetic interaction analysis between ⌬ste20 or ⌬gad8 mutant cells and ⌬brc1 revealed that the double mutant cells are viable but show additive effects with respect to sensitivity to MMS or CPT (Fig.…”

“…The presence of Rad52 foci may represent defects in DNA replication (e.g. incompletion of replication, as has been suggested previously (52). The number of Rad52 foci is elevated to very high levels in TORC2-Gad8 mutant cells in response to replication stress (Fig.…”

“…Similar results were obtained from studies of Brc1, a six-BRCT domain protein (52). ⌬brc1 cells show elevated levels of spontaneous Rad52-YFP foci (53) but reduced spontaneous recombination rates between direct repeats (52).…”

“…⌬brc1 mutant cells are sensitive to prolonged but not acute exposure to replication stressors and show an elevated level of spontaneous Rad52-YFP foci, which, as in TORC2 mutant cells, are not associated with activation of Chk1. In addition, as in TORC2 mutant cells, spontaneous recombination rates are decreased in ⌬brc1 mutant cells (52). As suggested above, Rad52 foci may not represent sites of recombination but incompletion of replication under conditions of replication stress.…”

Brc1 links replication stress response and centromere function

“…[19][20][21] However, a recent study indicated that the requirement for Brc1 more likely involves a role in the resumption of DNA replication at stalled or damaged replication forks. 22 In the case of collapsed replication forks, resumption of DNA replication absolutely depends on homology directed repair. 47 The one-ended DSB formed by the collapsed replication fork invades the sister chromatid to re-establish the replication fork.…”