During DNA replication, the extension of daughter strands is continuously impaired by a number of factors, such as proteins bound to the template, endogenously or exogenously induced DNA damage, and the presence of DNA secondary structures. If the replication fork stalls, and if the stalled fork is not processed to restore fork progression, disassembly of the replication complex can ensue. The stalled forks can also break, generating a double-strand break (DSB). Additionally, the presence of a DNA lesion, such as a single-strand nick in the template strand, can lead to a DSB. Consequently, a failure to repair the replication-associated lesions, and to then restart the stalled fork, will lead to chromosome loss or impairment of the integrity of the genome. Maintenance of the stability of the genome is critical for normal cell growth and cell viability.
To avoid genetic instability, cells have evolved a variety of mechanisms to rescue the stalled fork; extensive studies in both prokaryotes and eukaryotes suggest that homologous recombination plays a critical role in repair of the replication-associated DNA lesions, and in allowing the replication to continue [1–3]. For example, DSBs arising as a result of replication defects can be repaired by homologous recombination, using the sister chromatid as a template. Similarly, a replication fork stalled due to the presence of a replication block can be reinitiated by a template-switching mechanism, before the replication block is removed. However, unscheduled recombination can be detrimental, leading to a higher rate of genetic instability, as observed in the cancer-prone Bloom, Werner, and Rothmund-Thomson syndromes, respectively due to mutations in the BLM, WRN, and RECQL4 genes . These three genes belong to a highly conserved family of RecQ DNA helicases, originally described in Escherichia coli as a component of the RecF recombination pathway [4, 5].
BLM cells show a high rate of sister-chromatid exchange (SCE), and the sensitivity of both BLM and WRN cells to S-phase-specific inhibitors (e.g., camptothecin) suggests that these genes function during DNA replication . In addition, there is mounting evidence in yeast suggesting that replication does not proceed normally in the absence of RecQ helicases. Cells lacking the RecQ homolog Sgs1 in Saccharomyces cerevisiae exhibit an increased sensitivity to DNA-damaging agents (e.g., ultraviolet light, hydroxyurea, and methyl-methane sulphonate); an increased level of recombination between homologous sequences and between modestly divergent DNA sequences; gross chromosomal rearrangements; unequal SCE; and mitotic chromosome non-disjunction [6–12]. The Sgs1 protein closely associates with the replication fork and is thought to stabilize and restart the stalled fork [13–15]. In vitro studies have indicated that Sgs1, like its human counterpart, is a 3'-5' DNA helicase that can disrupt a variety of DNA structures, including cruciform structures that resemble the Holliday junction intermediate of the recombination process, suggesting its possible role in homologous recombination . Sgs1 physically interacts with type I topoisomerase I (Top3), and both genetic and biochemical studies indicate that the Sgs1/Top3 complex acts on Holliday junctions to suppress crossover outcomes [17–20].
Several synthetic lethal screens have been employed to identify the genes that are functionally related to Sgs1 [21–27]. The sgs1 mutation is synthetically lethal with a mutation in the SRS2 gene, which encodes another 3'-5' DNA helicase . Cells lacking Sgs1 and Srs2 are extremely sick, and the growth defect is suppressed by a mutation in any of the RAD51, RAD52, RAD55, and RAD57 genes involved in early stages of homologous recombination [21, 24, 27, 28]. Since the Sgs1 and Srs2 proteins function during DNA replication [4, 29], it has been proposed that Sgs1 and Srs2 in wild-type cells regulate the accumulation of toxic recombination intermediates, during DNA replication. In vitro studies have shown that Srs2 possesses an anti-recombination activity; it displaces Rad51, a strand-annealing protein, from DNA filaments [30, 31], which is in agreement with Srs2's in vivo recombination-inhibiting activity .
The sgs1 mutation is also synthetically lethal with mus81, but a rad51 mutation suppresses the lethal effect of the double mutation [22, 24], suggesting that Sgs1 and Mus81 function in separate pathways. Mus81 acts in a complex with Mms4; the heterodimeric protein has been shown to cleave branched DNAs and has been implicated in DNA repair; it also functions during sporulation [33–35]. Results of several genetic studies have led to the proposal that DNA structures formed during replication are acted upon by recombination proteins, forming intermediates that are toxic unless processed further. Sgs1/Top3 and Mus81 are needed to process these intermediates, whereas Srs2 limits the numbers of such intermediates, by disrupting Rad51 filaments.
In eukaryotes, SCE occurs spontaneously, probably representing recombination events during replication. The factors that impair the normal progression of the replication fork are likely to increase the rate of spontaneous SCE. One of the factors that compromises the normal progression of the replication fork is the presence of inverted repeats (IRs) that can form secondary structures in single-stranded DNA, by intra-strand base pairing between complementary sequences. Consistently, IRs have been found to be associated with gross chromosomal rearrangements [36, 37]. Previously, we constructed a recombination substrate to study the effect of IRs on unequal SCE in haploid S. cerevisiae . The presence of the repeated sequences increases spontaneous unequal SCE by about 10-fold [38, 39]. While non-IR-mediated SCE events are independent of DSB-repair genes, IR-stimulated SCE events depend on DSB repair genes, suggesting that IR-associated SCEs occur by DSB repair .
During DNA replication, the lagging strand is expected to contain a higher level of single-stranded regions than does the leading strand, due to the discontinuous nature of DNA synthesis. The single-stranded regions facilitate the formation of a secondary structure at an IR. The secondary structures are also substrates for structure-specific nucleases in vivo. Cleavage of the secondary structure at the stalled fork will lead to the formation of a DSB that can be repaired by either gene conversion or break-induced replication , using the sister chromatid as a template. In the present study, we analyzed IR-stimulated unequal SCE in cells lacking Sgs1 and/or functionally related enzymes that are believed to function at the stalled replication forks. Our results showed that the sgs1 mutation increases unequal SCE for both IR-containing and non-IR-containing substrates. However, IR-stimulated SCE events in the sgs1 background are significantly reduced when defects in the mismatch repair (MMR) gene MSH2 are also present. Additionally, we showed that SGS1 and EXO1 regulate SCE in two distinct pathways.