RNH lacking cells suffer from premature S-phase entry
R- loop formation partially overcomes cdc7-4 temperature sensitivity
The initiation of DNA replication in eukaryotic cells during S phase is regulated by 'origin licensing', and requires the sequential assembly of pre-RC proteins, such as Cdc7 and Cdc6, at the ARS. Cdc6 is an essential component of the pre-RC and acts prior to Cdc7 kinase activation, loading the Mcm2-7 proteins onto the ORC (239,240). Firing from normally inactive origins, or the formation of entirely new sites of replication initiation could account for the completion of DNA synthesis more quickly in the absence of RNaseH activity. We questioned whether the origin-independent, R-loop-mediated replication initiation (see Chapter 1), could by-pass the need for canonical origin firing. To address this question we created a cdc7-4 rnh1∆ rnh2∆ triple mutant (Figure 28A). The cdc7-4 allele permits growth at 23ºC (permissive temperature) but not at 30ºC (4) because the Cdc7 kinase is essential for the opening up and firing of replication origins by phosphorylation of Mcm2-7 proteins (4,241). Notably, restrictive temperature was shifted from 30°C to 37ºC in the cdc7-4 rnh1∆ rnh2∆ triple mutant, indicating that R-loops could help to make replication origins more accessible to replication factors. Additionally, the CPT sensitivity of cdc7-4 rnh1∆ rnh2∆ mutants was comparable to
rnh1∆ rnh2∆ mutants at 23ºC, but this sensitivity was increased dramatically at 30ºC (Figure 28B). Interestingly, transcription through origins of replication has been shown to inactivate replication firing (242). It is conceivable, that R-loop stabilization within replication origins by CPT could have the same effect.
Figure 28. R-loop formation partially overcomes cdc7-4 temperature sensitivity.A. Viability
of cdc7-4 simple and triple mutants grown at 23º, 30º, or 37ºC. B.Drop test analysis of cdc7-4
rnh1∆ rnh2∆ at 23º or 30º on YPAD or YPAD-containing CPT (5µg/ml). C. Tetrads (top) and drop
test analysis for cdc7∆ mcm5-bob1 quadruple mutant. Plates contained 0.5 or 1µg/ml CPT.
Besides its well-reported roles in replication initiation, Cdc7 also operates in post-replicative repair (PRR). Cdc7 is a member of the DNA damage tolerance RAD6 epistasis group, associated with the TLS branch (243). It has been proposed that different cdc7 alleles can result in hyper- or hypo-mutagenic phenotypes (244). To exclude the possibility that the PRR function of Cdc7 was interfering in the analysis of viability of the cdc7-4 rnh1∆ rnh2∆ mutant we opted to use a
cdc7∆ mcm5-bob1 strain. The function of Cdc7 in replication initiation is no longer essential in
a mcm5-bob1 (P83L) mutant (245), since presence of the mcm5-bob1 allele bypasses the need for the phosphorylation and activation of the MCM helicase by Cdc7 kinase (246). Analysing the viable spores from genetic crosses between the cdc7∆ mcm5-bob1 and rnh1∆ rnh2∆ yeast strains, we attained cdc7∆ mcm5-bob1 rnh1∆ rnh2∆ quadruple mutant spores (Figure 28B).
We confirmed the mcm5-bob1 genotype by back-crossing the quadruple spore with a WT yeast strain: the ability to recover viable cdc7∆ spores from the back-cross meant that the yeast also carried the mcm5-bob1 allele, originating from the quadruple spore from the first cross (results not shown). These analyses tell us that cdc7∆ rnh1∆ rnh2∆ yeast are inviable without the compensatory mcm5-bob1 allele, and that Cdc7 kinase activity is essential in the RNH- mutant,
as for the WT. Therefore, the origin-independent transcription-initiated replication events that we observe in the RNH double mutant do not bypass the need for normal replication initiation from origins in S-phase.
RNH
-mutants are not held in G2/M in the absence of Mrc1 activity
The synthetic lethal interaction of RNH- mutants and SIC1 suggested that cells may suffer from
constrains that may be generated during replication and still be present when cells are in the next G1 phase. To assess for replication-associated DNA damage, we inactivated different S- phase specific checkpoint pathways including rad9, chk1, tel1, and rad24. No significant differences were observed in the cell cycle progression of the triple mutants tested; all the triple mutants exhibited a similar holding of cells in G2/M phase as per the RNH double mutant (data not shown), although they showed an additive CPT sensitivity (see Chapter 2, Table 1). Interestingly, we detected a synthetic lethal interaction of RNH- mutants with MRC1 (see
Chapter 2; Figure 24). Mrc1, mediator of the replication checkpoint, the homologue of human Claspin, associates with replication forks shortly after replication initiation, remaining during elongation as an integral part of the replication machinery (224) and has been shown to have a dual role, acting both as a component of the RF and as a mediator of the S/G2 checkpoint (247). Viable mrc∆ rnh1∆ rnh2∆ triple mutants notably manifested a different cell cycle progression profile to that of the RNH double mutant. The mrc1Δ rnh1Δ rnh2Δ triple mutant was able to re- enter G1-phase of the following cell cycle whilst the RNH double mutant remained held in late S/G2 (Figure 29A). Moreover, the mrc1Δ rnh1Δ rnh2Δ yeast exhibited an even more accelerated entry into and passage through the S-phase, compared to the RNH double mutant.
Furthermore, of significant interest was the finding that Mrc1 is important for cell survival in the absence of RNaseH enzymes. From tetrad analysis we detected that only 33% of triple spores were viable (Figure 29B). Remaining viable triple mutant spores have a growth defect, and interestingly, whilst the mrc1Δ itself is not CPT sensitive, elimination of MRC1 in the RNH double mutant resulted in a highly CPT sensitive triple mutant (Figure 29C). These results suggest that Mrc1 activity is crucial in cells lacking RNaseH activity and can protect them from CPT-induced replication stress.
Figure 29. Mrc1 protects cells from CPT-induced replication stress. A. Flow cytometry
analysis of mrc1 triple mutant cells grown in the presence or absence of CPT following release from
α-factor. B. Tetrad analyses of Mrc1 separation-of-function alleles (top) and Mrc1 mediator
complex members (bottom). C. Drop test analysis of Mrc1 separation-of-function alleles (top) and
Mrc1 mediator complex members (bottom). D. Flow cytometry analysis of Mrc1 mediator complex
members.
Since mrc1∆ rnh1∆ rnh2∆ triple mutants did not arrest in late S/G2-phase in response to CPT (Figure 29A), their increased CPT sensitivity could reflect a problem in S/G2-dependent damage repair or RF stability. To discriminate between these possibilities, we took advantage of the Mrc1 separation of function mutants. Combination of the checkpoint-defective allele
mrc1AQ,which cannot be phosphorylated by the checkpoint kinases (248), and the RF stability
sensitivity. These results suggest that both S/G2-checkpoint and RF stability functions of Mrc1 contribute to CPT tolerance in the rnh1Δ rnh2Δ background. In contrast, only the triple mutant with the RF stability mutant allele, mrc1-c14, closely resembled the growth, CPT sensitivity and spore viability phenotypes of the mrc1∆ rnh1∆ rnh2∆ mutant (Figures 29B and 29C). The
mrc1-c14 triple mutant spores were affected negatively in growth with some lethality observed
in meiotic segregants (87% viability of spores corresponding to triple mutants). Collectively, these results demonstrate that Mrc1 plays an important role in the stabilization of RFs in the
RNH- mutant, and may be particularly important for the survival of CPT-induced lesions, and
that both functions of Mrc1 are indispensable for the viability of yeast lacking RNaseH activity. Mrc1 exists as an integral member of the replisome (224), with Csm3 and Tof1, as part of the Mrc1-mediator complex. Csm3 and Tof1 are specifically required for the association of Mrc1 with the RF, interacting directly with the MCM helicase, and thus play a central role in RF progression. The complex is also important in maintaining the stability of stalled RFs and promoting subsequent DNA repair events (224,247). Genetic interaction analysis revealed novel functional relationships between the RNaseH enzymes and all members of the Mrc1 mediator complex (Figure 29B). Triple mutants of rnh1Δ rnh2Δ with csm3∆ and tof1∆ were synthetic sick; triple mutants also exhibited extreme sensitivity to CPT, in contrast to the CPT resistant csm3∆ and tof1∆ single mutants (Figure 29C). Cell cycle progression of the tof1∆ triple mutant, upon release from G1 synchronization into media in the presence of CPT, was very similar to that previously observed for the mrc1∆ triple mutant (Figure 29D). In contrast, the csm3∆ triple mutant remained held in late S/G2, as for the RNH double mutant, suggesting a possible difference in functions for individual members of the complex in the absence of RNaseH activity. These results indicate that the replication fork stabilization function of Mrc1 (together with Tof1) is important in protecting cells from R-loop mediated replication constrains and, similar to its role in the response to osmostress (250), support a role for Mrc1 in the co-ordination of transcription and replication events in CPT treated RNH- mutants.