The Fission Yeast Rad32(Mre11)–Rad50–Nbs1 Complex Acts Both Upstream and Downstream of Checkpoint Signaling in the S-Phase DNA Damage Checkpoint

Abstract

The Mre11-Rad50-Nbs1 (MRN) heterotrimer plays various and complex roles in DNA damage repair and checkpoint signaling. Its role in activating Ataxia-Telangiectasia Mutated (ATM), the central checkpoint kinase in the metazoan double-strand break response, has been well studied. However, its function in the checkpoint independent of ATM activation, as well as functions that are completely checkpoint independent, are less well understood. In fission yeast, DNA damage checkpoint signaling requires Rad3, the homolog of the ATR (ATM and Rad3-related) kinase, not Tel1, the ATM homolog, allowing us to dissect MRN's ATM-independent S-phase DNA damage checkpoint roles from its role in ATM activation. We find that MRN is involved in Rad3 (ATR)-dependent checkpoint signaling in S phase, but not G2, suggesting that MRN is involved in ATR activation through its role in replication fork metabolism. In addition, we define a role for MRN in the S-phase DNA damage checkpoint-dependent slowing of replication that is independent of its role in checkpoint signaling. Genetic interactions between MRN and Rhp51, the fission yeast Rad51 homolog, lead us to suggest that MRN participates in checkpoint-dependent replication slowing through negative regulation of recombination.

Role for the Mammalian Swi5-Sfr1 Complex in DNA Strand Break Repair through Homologous Recombination

“…For the most part, the sensitivities of Swi5 −/− and Sfr1 −/− cells are similar to each other, although unlike Swi5 −/− cells, Sfr1 −/− cells are also sensitive to etoposide. In contrast to Swi5, the stability of Sfr1 is not fully compromised when its partner protein is absent, consistent with Sfr1 functions that are independent of Swi5 in some contexts, as is the case with fission yeast [52] , [53] . While fission yeast Swi5 acts independent of Sfr1 during mating-type switching, mouse Swi5 is unlikely to have Sfr1-independent functions, given its instability in the absence of Sfr1.…”

Initiation of DNA damage responses through XPG-related nucleases

“…Additional resection is carried out through multiple different pathways including those utilizing RecQ helicases, and the nucleases, Exo1 and Dna2 (Nakada et al , 2004; Limbo et al , 2007; Sartori et al , 2007; Gravel et al , 2008; Liao et al , 2008; Mimitou and Symington, 2008; Zhu et al , 2008; Mimitou and Symington, 2009). Defects in these genes impacts on recombination, however, Chk1 activation still occurs in the absence of MRN (Grenon et al , 2001; Willis and Rhind, 2010; Limbo et al , 2011), Ctp1 (Limbo et al , 2007; Sartori et al , 2007) and Exo1 (Gravel et al , 2008 and see below) suggesting other mechanisms of checkpoint activation must exist.…”

Preserving the Replication Fork in Response to Nucleotide Starvation: Evading the Replication Fork Collapse Point

“…Intriguingly, MRN/MRX-components co-localize at the replication fork and are important for fork stability (e.g. [35,149,[160][161][162]), pointing at the essential role of recombination repair in restarting stalled replication forks.…”