Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

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The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo . Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

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  1. Author Response:

    Reviewer #3:


    • The in vivo suppression phenotype is relatively minor and suggests that other factors or pathways play a significant role in vivo. For example, the Diffley lab has previously shown that deletion of EXO1 almost completely suppresses the drug sensitivity of rad53∆ cells. This suggests that the Rad53-Mrc1 axis of regulation described here remains insufficient to prevent the accumulation of Exo1-sensitive lesions or conditions.

    We agree that the MRC18D rescue of the rad53Δ sensitivity to HU and MMS is small. We note, however, that it is reproducible and suggest that it may be only one part of Rad53’s complex role in protecting replication forks during the replication checkpoint. Because Mcm10 cannot be phosphorylated in MRC18D rad53Δ cells, replication speed is likely to be faster than in RAD53 wild-type cells, where both Mrc1 and Mcm10 would be phosphorylated. Thus MRC18D by itself may not provide sufficient fork slowing to protect forks. Though outside the scope of this work, it would be interesting to identify an Mcm10 mutant to combine with the Mrc1 mutant and see if this further rescues rad53Δ sensitivity. We have added this point to the discussion on page 14. We also note that the ability of EXO1 deletion to suppress drug sensitivity has turned out to be more complex than originally believed because of suppressors which accumulate rapidly in rad53 mutants (Gómez-González et al, Genetics, 2019), hence the importance of tackling this issue with biochemistry.

    • Despite the development of a quantitative method for DNA unwinding and the high quality of the data, there is no quantitative analysis of the data by statistical method. At least there needs to be clear evidence of reproducibility.

    We have now included the details of how we quantify the unwound DNA in the helicase assay and how we extract the unwinding rate to the Experimental Procedures “CMG helicase assay” section on page 17. We have also added the 95% confidence interval for the unwinding rate in the “Mrc1 regulation of replication rate” section of the results on page 8.

  2. Evaluation Summary:

    This manuscript will be of interest to those interested in the regulation of DNA replication and those interested in how DNA damage impacts DNA replication. DNA replication must occur accurately to maintain genome integrity and also must be able to deal with DNA damage or metabolic conditions that induce replication fork stalling. Two key proteins involved in signaling such replication stress are Mrc1 and Rad53 kinase, and the authors use a powerful in vitro reconstitution system to make findings pertaining to these two proteins that are then supported by genetics.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

  3. Reviewer #1 (Public Review):

    This paper addresses the role of the DNA damage and DNA replication stress proteins Mrc1 and Rad53 kinase in DNA replication fork progression. The authors confirm previous research that Rad53 can block the initiation of DNA replication by phosphorylating either Dbf4 or Sld3. They also show that Mrc1 stimulates the movement of the CMG helicase on a n activated dsDNA substrate and that Rad53 phosphorylation of Mrc1 blocks this helicase stimulation and thus DNA replication fork progression. They also show that Rad53 can target Mcm10, but the consequences of this phosphorylation are only revealed in the absence of Mrc1. The authors confirm the effect of the Mrc1 phosphorylation mutations in vivo.

    This paper reports new functions for the Rad53 checkpoint kinase and for Mcm10 and Mrc1 at DNA replication forks. The results have important implications for understanding the response of the DNA replication machinery to DNA damage and the separation of this activity from global checkpoint signaling.

  4. Reviewer #2 (Public Review):

    In this manuscript, McClure and Diffley take advantage of their powerful in vitro reconstitution system of budding yeast DNA replication to explore the role of Rad53, a key DNA damage signaling kinase, in the slowdown of DNA replication progression. They identify two potential Rad53 targets: Mrc1 and MCM10 and show that Mrc1 phosphorylation is both essential and sufficient for slowing down DNA replication by slowing down the unwinding rate of the replicative helicase.

    Overall, this manuscript makes some very useful contributions to our understanding of the control of DNA replication progression: it identifies Mrc1 as a key and direct Rad53 target in the replisome and it also provides the basis of Mrc1-dependent inhibition of replisome progression via slowing down the unwinding rate of the replicative helicase. While the results are broadly supportive of their conclusions, the work would be strengthened if the authors could provide additional evidence that the sites on Mrc1 they identify as being critical for slowing down replisome progression in vitro, are also phosphorylated by Rad53 in yeast cells.

  5. Reviewer #3 (Public Review):

    Background and Significance:

    DNA replication of in the presence of DNA damage or nucleotide depletion results in activation of the DNA checkpoint signaling pathways that trigger numerous responses to minimize genome damage. The signaling pathway results in the Mec1/ATR kinase-dependent phospho-activation of the Rad53 effector kinase that in turn targets multiple replication and DNA repair factors to maintain the integrity of the replication process. Previous in vivo studies have determined that Rad53 responds to replication stress by inhibiting the activation of additional, unfired replication origins, and by stabilizing replication forks to prevent their collapse into dsDNA breaks or other destabilizing structures. Previous studies have also indicated that fork stabilization rather than origin inhibition is the critical essential function of Rad53 in maintaining genome stability and cellular viability. However, exactly how Rad53 activity stabilizes forks remains incompletely understood. Some in vivo studies have suggested that Rad53 slows the progression of the replicating fork(s), perhaps to provide time for repair of template damage or restoration of dNTP levels before attempted replication. However, the effect of Rad53 on fork rate has been somewhat controversial, and critical targets of Rad53 at the fork required for fork stabilization and fork rate control have remained obscure despite much interest in identifying these factors, which are considered to be central to genome stability.

    Detailed Review:

    McClure and Diffley's current work reports on the function of Rad53 in regulating origin firing and particularly the elongation phase of replication. The authors use an in vitro replication system using step-wise addition of purified proteins to examine the effects of Rad53 activity in the absence of DNA damage or dNTP depletion, thereby eliminating these as confounding factors.

    The authors begin by loading MCMs onto a plasmid template, followed by DDK activity, which has been pre-incubated with active Rad53 kinase or mutant Rad53 as control. Consistent with expectations from previous in vivo studies, phosphorylation of DDK inhibits replication initiation in this system. Similar treatment of Sld3 by Rad53 similarly inhibits replication initiation as expected from previous in vivo work. The experiments indicate that Rad53-dependent phosphorylation of either Dbf4 or Sld3 is sufficient to inhibit replication initiation.

    Next, the authors examine the effect of exposing elongation factors to Rad53 following the execution of the initiation steps. This results in a reduction of elongation rate from ~700bp/min to ~400bp/min. The authors narrow the focus to Mrc1, Tof1, and Csm3, which together comprise a so-called replication fork protection complex, by specifically pre-incubating these proteins with Rad53. They find that pre-incubation of Mrc1 alone is sufficient to slow elongation whereas pre-treatment of Csm3 and Tof1 had little or no effect. They further showed that Rad53-mediated inhibition of elongation requires only Mrc1 phosphorylation, as add-back of Rad53-untreated Mrc1 to replication reaction with other elongation factors pre-treated with Rad53 restored normal elongation rate.

    In possible conflict with these results, the Remus lab has recently reported that Rad53-mediated phosphorylation of Mcm10 slows elongation in reconstituted replication reactions lacking Mrc1, Csm3 and Tof1 (M/C/T). The authors here confirm this result but show that the effect is only observed in the absence of M/C/T, and add back of untreated M/C/T rescues elongation rate to wild-type level. These results suggest that Mcm10 phosphorylation may regulate fork rate in concert with phosphorylated Mrc1, but not unphosphorylated Mrc1.

    The authors further investigated the mechanism of Mrc1 fork slowing in the presence of different replicative DNA polymerases, and find that slowing is independent of the DNA polymerase involved. To determine whether Mrc1 affects DNA unwinding, they developed an assay for unwinding based on DNA cleavage by a restriction endonuclease, which can only cut dsDNA. Thus, DNA unwinding can be measured by loss of cleavage at increasingly distal sites from the replication origin. In this assay, presence of Mrc1 stimulates the unwinding rate approximately two-fold, whereas phosphorylated Mrc1 does not, suggesting that Rad53 phosphorylation of Mrc1 inhibits its stimulatory effect of the helicase. It's notable that the rate of unwinding in this assay is roughly five-fold slower than progression of the synthesizing replication fork in this system. This, of course, suggests that additional elongation factor(s) contribute to the full rate of replication in this system.

    To dig deeper into mechanism of Mrc1 inhibition, the authors examine mutant alleles lacking numerous potential phosphorylation sites, starting with the 17AQ mutant, which lacks Mec1-target sites, and is defective in signal transduction to Rad53. Interestingly, this mutant has no effect on Rad53 inhibition. A C-terminal truncation of Mrc1 eliminated inhibition by Rad53; however, this allele also fails to stimulate replication like wild-type Mrc1, complicating interpretation. The authors take multiple approaches to map Rad53 phosphotargets in Mrc1, including incubation of Rad53 with Mrc1 protein fragments, with Mrc1 peptide arrays, and MS. Most of the Rad53-dependent sites are located to the C-terminus and the authors create multisite mutants (to alanine) to determine functional effects of eliminating phosphorylation. Although they failed to identify a perfect separation of function mutant that retained full stimulation of replication while eliminating inhibition by Rad53, the 14A and 19A mutants exhibit intermediate phenotypes for both stimulation and inhibition, consistent with these residues serving a regulatory function.

    They alternatively mutated a subset of the 14A residues to phosphomimics, creating an 8D allele. With or without phosphorylation by Rad53, the Mrc1-8D supports replication equivalently to phosphorylated wild-type Rad53 as predicted for this phosphomimicking allele. One concern, of course, is whether the residue changes have inactivated normal Mrc1 stimulation of replication independently of effective phosphomimics. The finding that 14A affects Mrc1 function in the absence of Rad53 contributes to this concern as mutation to A is expected to results in less potential pleiotropic effects than changes to D.

    The authors go on to test the effects of the 8D allele in vivo on response to DNA damaging (MMS) and nucleotide depleting (HU) agents. Intriguingly, they find that combination of the Mrc1-8D allele with rad53∆, partially suppresses the extreme drug sensitivity of rad53∆. This is an interesting finding that supports the idea that phosphorylation of Mrc1 by Rad53 contributes to survival in the presence of these damaging agents. Arguably, this may also support the idea that the 8D allele is not simply broken (comparison to mrc1∆ might help make that point though rad53∆ mrc1∆ may be lethal, which also supports this conclusion). Indeed, the 8D allele supports Rad53 activation in vivo, indicating that it retains some function. Overall, this is a compelling result despite the modest effect and lingering caveats about this allele.


    - Answers, at least partially, the long-standing question in the field: How does Mrc1 control replication forks normally and under replication stress? This has been challenging to address by in vivo experiments for multiple reasons including multiple pathways, targets, and use of drugs to elicit Rad53 activation, which has independent/additional effects on forks.

    - Given the complex role of Mrc1 in coordinating multiple activities at the replication fork, the highly defined biochemical system with purified proteins is appropriate to the task. Experiments are well-controlled and replication is robust. Many complicating effects are eliminated. Most results are clear-cut and convincing.

    - Identification of Rad53 phosphosites in Mrc1 and likely role of these sites in regulating the rate of fork progression is a substantial accomplishment and these results provide a defined mechanism to incorporate into existing models of Rad53 and replication control.


    - The in vivo suppression phenotype is relatively minor and suggests that other factors or pathways play a significant role in vivo. For example, the Diffley lab has previously shown that deletion of EXO1 almost completely suppresses the drug sensitivity of rad53∆ cells. This suggests that the Rad53-Mrc1 axis of regulation described here remains insufficient to prevent the accumulation of Exo1-sensitive lesions or conditions.

    - Despite the development of a quantitative method for DNA unwinding and the high quality of the data, there is no quantitative analysis of the data by statistical method. At least there needs to be clear evidence of reproducibility.