Academic journal article Genetics

Sgs1 and Mph1 Helicases Enforce the Recombination Execution Checkpoint during DNA Double-Strand Break Repair in Saccharomyces Cerevisiae

Academic journal article Genetics

Sgs1 and Mph1 Helicases Enforce the Recombination Execution Checkpoint during DNA Double-Strand Break Repair in Saccharomyces Cerevisiae

Article excerpt

DNA double-strand breaks (DSBs) are potentially lethal lesions that can be repaired either by nonhomologous end joining (NHEJ) or by homologous recombination (HR) (Krogh and Symington 2004; Haber 2008; Haber 2013). While NHEJ involves simple religation of the DSB ends with little or no homology, HR requires the presence of intact homologous sequences to serve as a template for repair. During HR-mediated repair, the DSB ends are resected to produce 39-ended single-stranded DNA tails, which get coated with the Rad51 recombinase protein to form Rad51 nucleoprotein filaments. These Rad51 filaments then search for and strand invade homologous sequences to form a three-strand displacement loop (D-loop), which is followed by extension of the invading strand by new DNA synthesis using the paired homologous sequence as a template. When both DSB ends share homology with a sister chromatid, a homologous chromosome or an ectopically placed donor; repair occurs by gene conversion (GC), resulting in the synthesis of a short patch of new DNA at the recipient locus using the homologous donor as a template. New DNA synthesis is initiated within 30 min after strand invasion (White and Haber 1990; Sugawara et al. 2003; Hicks et al. 2011) and does not require components of the lagging strand DNAsynthesis machinery such as Pola and primase (Wang et al. 2004). In mitotic cells of budding yeast, GC proceeds primarily by the synthesis-dependent strand annealing (SDSA) mechanism, in which the newly-synthesized strands dissociate from the donor template and are returned to the recipient locus. GC is thus distinct from normal semiconservative replication in that all the newly-copied DNA is found in the recipient (Ira et al. 2006).

However, if there is homology to only one DSB end, repair occurs by another HR pathway called break-induced replication (BIR) (McEachern and Haber 2006; Llorente et al. 2008; Malkova and Ira 2013). Here, strand invasion by the homologous end results in the establishment of a migrating D-loop that can copy all sequences distal to the site of homology, resulting in a nonreciprocal translocation (Donnianni and Symington 2013; Saini et al. 2013; Wilson et al. 2013). Sequences on the other side of the break, lacking homology, are lost by degradation.

Compared to GC, BIR is less efficient; it is kinetically slower and new DNA synthesis does not initiate until ^3-4 hr after strand invasion (Malkova et al. 2005; Lydeard et al. 2007; Jain et al. 2009). BIR requires leading and lagging strand DNA synthesis and essentially all of the DNA replication factors including Pola/primase, Cdc7, Cdt1, and the Cdc45MCM-GINS helicase complex (Lydeard et al. 2007; Lydeard et al. 2010b); only the components specifically needed for origin-dependent DNA replication (Cdc6 and the ORC proteins) are dispensable for BIR. Additionally, while Pold and Pole are redundantly required for GC (Holmes and Haber 1999; Wang et al. 2004), Pold alone is required for the initiation of BIR whereas Pole becomes important for the completion of repair (Lydeard et al. 2007).

In Saccharomyces cerevisiae, when both ends of a DSB share homology to the donor, GC nearly always outcompetes BIR (Malkova et al. 2005). Although GC is the more commonlyused mode of DSB repair, BIR is required for the maintenance of telomeres in telomerase-deficient cells (Lundblad and Blackburn 1993; Le et al. 1999; Teng et al. 2000; McEachern and Haber 2006; Lydeard et al. 2007). A single homologous end can also arise when a DSB occurs in the vicinity of a dispersed, repeated sequence such as a transposable element that is present at other chromosome locations (Malkova et al. 2001; VanHulle et al. 2007).

Correct channeling of the DSBs is crucial to ensure that repair occurs with high fidelity. Independent initiation of BIR events from DSB ends that could repair by GC will produce nonreciprocal translocations if the ends are repaired by using two nonallelic, repeated donor sequences; or cause aneuploidy if one end is repaired using a sister chromatid while the other end used a homologous chromosome for repair. …

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