Our analysis of the genetic interaction of THO with genes involved in replication, S-phase checkpoint, DNA repair, and chromatin remodeling revealed a synthetic growth defect in double null mutants of THO and S-phase checkpoint factors, such as the RFC-like and PCNA-like complexes. Under replicative stress, hyperrecombinant THO null mutants require a functional S-phase checkpoint for survival that is linked to the formation of R-loops. Thus, R-loop-forming hpr1Δ mutants activate the phosphorylation of the checkpoint effector kinase Rad53. This is accompanied by replication fork progression impairment at transcriptionally active endogenous chromosomal regions. Altogether, our results indicate that R-loops mediate the formation of a replication-dependent DNA damage structure that is sensed by the S-phase checkpoint, which becomes essential for the cell survival of THO mutants under replicative stress.
Double deficiencies of THO together with checkpoint factors, including S-phase checkpoint sensors such as the RFC-like factor Rad24, PCNA-like components Mec3 and Ddc1, and the checkpoint kinases Mec1 and Rad53, showed a synthetic growth defect, while double mutants of hpr1
Δ with mutations in canonical RFC and PCNA (cdc44
, respectively) grew normally (Fig. and ). The synthetic growth defect caused by THO null mutations in combination with mutations in the S-phase checkpoint factors is exacerbated in the presence of replicative stress, as tested by sensitivity to HU. Interestingly, this was not occurring with hpr1
), a nonhyperrecombinant mutation that does not form R-loops (23
), implying that the cotranscriptional formation of R-loops is needed for the S-phase checkpoint requirement. Consistently, Rad53 phosphorylation could be detected in hpr1
Δ cells (Fig. ) and hpr1
Δ increases the levels of the RNR3 ribonucleotide reductase (N. Proudfoot, personal communication), an indicator of the existence of a checkpoint response. These results suggest that the replication-born damaged DNA structures formed in hpr1
Δ cells are sufficient to activate the DNA damage checkpoint. We cannot disregard that the nontranscribed single-stranded DNA in the R-loop could be covered by RPA and sensed by the checkpoints, although we have no evidence for this possibility yet.
The primary event allowing checkpoint proteins to protect stalled forks appears to be replication fork stabilization (69
), and many replication proteins are targeted by the checkpoint machinery, probably to stabilize the replisome fork association when forks stall (41
). This is, for example, the case of the replicative primase, and indeed, mutations in the replicative primase (pri1-M4
) also gave rise to HU sensitivity in hpr1
Δ. Similarly, the requirement of ubiquitin-ligase Dia2 for the survival of hpr1
Δ cells under replication stress might be explained by the essential role of Dia2 for the stabilization of replication forks through regions of damaged DNA (12
). Therefore, it seems that R-loop-forming THO mutants need the genes involved in replication fork stabilization after DNA damage for survival. This is consistent with the replication fork impairment of THO mutants observed in the endogenous chromosome (Fig. ) and an artificial plasmid (71
) as well as the higher genetic instability observed in the absence of a functional S-phase checkpoint (Fig. ). The hyperrecombination of THO mutants is still observed (and further increased) in checkpoint-defective cells, consistent with the recent observation that the replication checkpoint does not affect homologous recombination at replication forks, but it impedes DSB repair (4
). It would certainly be interesting to decipher the putative roles of the S-phase checkpoint to overcome replication fork stalling in addition to its role in replisome stabilization. Notably, Hpr1 has been recently identified as a Mec1 target (46
), raising the intriguing possibility that THO could have a direct role in replication fork progression through transcribed DNA.
The S-phase checkpoint requires a threshold level of DNA damage for activation so that cells can tolerate a certain level of fork-associated ssDNA without leading to cell-cycle arrest. Later in the cell cycle, a threshold level is not required anymore, and the persistence of DSBs or regions of unreplicated DNA in the G2
phase is enough to prevent progression into mitosis (62
). The observation that hpr1
Δ cells tend to arrest in G2
suggests that this arrest must be the cause of their HU and MMS sensitivity. Supporting this, HU sensitivity is reversible in these mutants and can be suppressed by the deletion of the mitotic checkpoint gene MAD2
, implying that a Mad2-mediated checkpoint detected the damaged replicative structure generated in the double mutants in THO and checkpoint factor cells under replicative stress (Fig. ).
Taken as a whole, our data suggest that THO mutants accumulate damaged DNA structures during replication that are sensed by the S-phase checkpoints. The emerging questions are, what are these replication-born damaged DNA structures, and how are they repaired? We propose that these structures could be ssDNA gaps, since double mutants harboring null mutations of a THO gene and a DSB repair gene are viable, even in the presence of replicative stress. Instead, a number of mutants that have been either proposed or demonstrated to accumulate DSBs are not viable when harboring null mutations in DSB repair genes. This is the case with rad27
Δ or srs2
Δ cells harboring mutations in the genes encoding the MRX complex (70
). Cotranscriptionally formed RNA-DNA hybrids in THO mutants may cause replication fork stalling on either the leading or lagging strand with the concomitant accumulation of ssDNA that can be covered by RPA and sensed by the S-phase checkpoints (Fig. ). Presumably, these RNA-DNA hybrids would be efficiently removed or bypassed by a still-unknown mechanism that may involve ribonucleases or some putative DNA-RNA helicases. Alternatively, in codirectional collisions, the replisome could use the RNA strand of the R-loop as a primer and displace the RNAPII, as it has been recently shown for in vitro leading-strand replication in Escherichia coli
). This would also lead to the accumulation of ssDNA behind the fork that would need to be filled by postreplicative repair. However, when this stalling occurs within a DNA-direct repeat region, the hybrid could be bypassed by recombination, thus explaining the strong hyperrecombination between direct repeats in THO mutants.
FIG. 7. Possible consequences of replication through cotranscriptional R-loops. RNA-DNA hybrids occurring on either the leading or the lagging strand might cause replication fork stalling with the concomitant accumulation of ssDNA, which is sensed by the S-phase (more ...)
It is generally assumed that mitotic recombination is initiated by DSBs, but it has not been discarded that other lesions, such as ssDNA gaps, could initiate homologous recombination. Indeed, there is evidence supporting that spontaneous mitotic recombination, such as that occurring at stalled replication forks, may not necessarily be initiated by DSBs (16
). Based on the genetic requirements of hpr1
Δ viability in the presence of replicative stress, including S-phase checkpoint factors but not DSB repair factors, as discussed above, it would be possible that the hyperrecombination of THO mutants is not primarily initiated by DSBs. In favor of this idea is the fact that the hyperrecombination of THO mutants is almost restricted to direct repeat recombination (2
) or that chromosomal rearrangements are poorly stimulated (5- to 10-fold [Fig. ]) compared to the increase in direct repeat recombination of 3 orders of magnitude. Our study opens the intriguing possibility that R-loops may not be sufficient for DSB formation, which would explain why R-loop-mediated class switching in B cells requires AID as an essential player in the formation of DSBs (44
), and indicates that the genomic instability caused by R-loops is mediated by replication.
We know from bacterial studies that homologous recombination is critical for replication fork restart after its blockage and collapse. However, homologous recombination is also involved in lesion bypass (reviewed in reference 26
). In the latter case, it has been speculated that homologous recombination can be initiated by template switching between the nascent DNA strands, which would be stimulated by the ssDNA gaps that arise as a consequence of replication fork stalling (see reference 25
). Therefore, it seems plausible that the RNA-DNA hybrid occurring within DNA repeats could be bypassed by a recombination-mediated replication process involving inter- or intramolecular template switching. Indeed, such a kind of template-switching model at replication forks has been suggested in mammals to explain the bypass of the DNA polymerase over the transcription machinery (60
Interestingly, the hpr1
, and hpr1
Δ double mutants grow poorly under replication stress. This indicates that 5′ resection may be required to allow recombination-mediated template switching, since Dna2 has recently been shown to have a role in DSB resection together with Sgs1 (73
). Another certainly interesting observation was the stronger HU sensitivity of the hpr1
Δ double mutant. Pol32 is the only nonessential subunit of Pol δ. In addition to its role in Okazaki fragment maturation, Pol32 is involved in replication restart via the recombination-mediated replication mechanism break-induced replication (43
). Pol32 could be required for recombination-mediated replication between DNA repeats. Alternatively, Pol32 could be required for replication through a DNA template hybridized with RNA. Interestingly, in vitro studies have shown that RNA-DNA hybrids are displaced by Pol δ at higher efficiency than DNA duplexes and that Pol32 is required to extend short flaps into very long flaps (66
). Either way, our result opens the possibility that Pol32 could be necessary for replication resumption or for the bypassing of obstacles by Pol δ.
In summary, our results indicate that in THO mutants, cotranscriptionally formed R-loops impair replication fork progression, generating the accumulation of ssDNA gaps that trigger the activation of S-phase checkpoints to stabilize the replisome. If this occurs within DNA direct repeats, the RNA-DNA hybrid can be bypassed by a recombination-mediated replication process involving inter- or intramolecular template switching, providing a new perspective on our understanding of the initiation of mitotic recombination and the way cells can bypass replication obstacles.