Mrc1 and DNA polymerase ε function together in linking DNAreplication and the S phase checkpoint

Huiqiang Lou1, Makiko Komata2, Yuki Katou2, Zhiyun Guan1, Clara C. Reis1, Martin Budd1,Katsuhiko Shirahige2, and Judith L. Campbell1,*

1 Braun Laboratories, California Institute of Technology, Pasadena, California 91125

2 Laboratory of Genome Structure and Function, Division for Gene Research, Center for BiologicalResources and Informatics, Tokyo Institute of Technology, B-65, 4259, Nagatsuta, Midori-ku, Yokohama City,Kanagawa, 226-8501, Japan

SUMMARYYeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replicationforks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalyticsubunit of DNA polymerase ε, essential for leading strand DNA replication and for the checkpoint.In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halvesof Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpointabolishes Pol2N binding but not Pol2C interaction. Mrc1 is required to stabilize Pol2 at replicationforks stalled in HU. The bimodal Mrc1/Pol2 interaction may identify a novel step in regulating theS phase checkpoint response to DNA damage on the leading strand. We propose that Mrc1, whichalso interacts with the MCMs, may modulate coupling of polymerization and unwinding at thereplication fork.

INTRODUCTIONTo achieve genome stability, complicated replication machines must be sequentially assembledand precisely programmed such that S phase is appropriately integrated into the cell cycle.Assembly and programming occur during the M/G1 phases of the cell cycle. In addition, thereplication machines must be protected during S phase, because progress of the replicationmachine along the template, while sequential, is not continuous. Replication forks stallperiodically, either in difficult-to-replicate regions of the genome or due to exogenousenvironmental interference. How to prevent replication forks from irreversibly collapsing in Sphase, when de novo assembly is no longer permitted, becomes a fascinating question. Cellsmust have a mechanism to stabilize the replication machinery in situ until aberrant structurescan be removed and forks can restart. The simplest solution is for this mechanism to be intrinsicto the replication machine itself. The studies described here comprise a genetic and biochemicalanalysis of one of these components, yeast Mrc1.

Mrc1 (mediator of the replication checkpoint) plays roles in both the S phase checkpoint, amultistep response to replication stress, and at the replication fork. First, phosphorylation ofMrc1 by the “signaling” Mec1/Ddc2 kinase is required for activation of the Rad53 “effector”

*Correspondence: E-mail: jcampbel@its.caltech.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptMol Cell. Author manuscript; available in PMC 2009 October 10.

Published in final edited form as:Mol Cell. 2008 October 10; 32(1): 106–117. doi:10.1016/j.molcel.2008.08.020.

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kinase during the S phase checkpoint (Alcasabas et al., 2001; Osborn and Elledge, 2003).Second, Mrc1, although it is not essential, is required for normal DNA replication in the absencereplication stress. Mrc1 is loaded onto replication origins in every cell cycle at the same timeas DNA polymerases, just after formation of the Replication Progression Complex, whose coreconsists of Cdc45, GINS, and the MCMs and migrates away from origins with replication forks(Alcasabas et al., 2001; Bjergbaek et al., 2005; Gambus et al., 2006; Katou et al., 2003; Szyjkaet al., 2005). Mrc1 plays an intrinsic and constitutive role in replication, since in its absenceall replication forks move at only half the normal rate and cells experience extensive replicationfork damage in the absence of exogenous damaging agents (Alcasabas et al., 2001; Azvolinskyet al., 2006; Bjergbaek et al., 2005; Osborn and Elledge, 2003; Szyjka et al., 2005). mrc1 nullsthus exhibit a high frequency of gross chromosomal rearrangements (GCRs) and syntheticlethality with rad9 null, encoding the DNA damage checkpoint “mediator” (Bjergbaek et al.,2005; Osborn and Elledge, 2003). Several essential proteins, pol ε, Dpb11, RFC5, and Cdc7/Dbf4, like Mrc1, also perform roles in both DNA replication and in the S phase checkpoint(Araki et al., 1995; Navas et al., 1995; Sugimoto et al., 1997; Sugimoto et al., 1996). Third,Mrc1 may participate in sister chromatid cohesion and telomere capping (Tsolou and Lydall,2007; Xu et al., 2004).

mrc1AQ, a mutant in which all of the possible Mec1 S/TQ phosphorylation sites have beenchanged to nonphosphorylatable AQ, is proficient in DNA replication but deficient in fullRad53 activation, and thus in the downstream global cell cycle responses of the checkpoint(Alcasabas et al., 2001; Osborn and Elledge, 2003). This separation of function mutant leadsto the notion that the replication and checkpoint functions of Mrc1 are separable. The situationmay be more complicated than that, however. When normal cells are transiently exposed toHU (hydroxyurea), forks stall, but the replisome, along with Mrc1, remains at the forks, whichcan restart when HU is removed (Bjergbaek et al., 2005; Katou et al., 2003; Szyjka et al.,2005; Tourriere et al., 2005). In an mrc1 null, however, a majority of the stalled forks fail torestart (Bjergbaek et al., 2005; Szyjka et al., 2005; Tourriere et al., 2005). This failure to restartforks correlates with an uncoupling of DNA synthesis and replisome migration (Katou et al.,2003). DNA synthesis ceases; but the replisome, including DNA polymerases α and Dpb3,Cdc45, the MCMs, and GINS, continues to translocate for thousands of base pairs withoutsynthesizing new DNA (Bjergbaek et al., 2005; Katou et al., 2003). Mrc1 is also a componentof forks paused at natural replication barriers formed by stable protein-DNA complexes, forinstance in the rDNA in yeast, along with DNA polymerases α and ε, Cdc45, the MCMS, andGINS (Azvolinsky et al., 2006; Calzada et al., 2005). Thus, Mrc1 participates directly in forkstabilization during the checkpoint cascade.

One mechanism by which Mrc1 might prevent uncoupling of synthesis and unwinding andstabilize the replisome at the stalled forks is by inhibiting progression of the Cdc45/MCMhelicase motor in the absence of a functioning polymerase motor. In partial support of thismodel, Mrc1 coimmunoprecipitates with Cdc45 (Katou et al., 2003). Mrc1 also copurifies,albeit in substoichiometric amounts, with the RPC (Gambus et al., 2006). In addition, in S.pombe, certain loss of function mutations in the Cdc45 and MCM orthologs suppress the HUsensitivity of mrc1Δ mutants (Nitani et al., 2006). However, MCM inhibition would not accountfor the fact that Mrc1 is a positive factor for normal DNA replication. Therefore, a morecomprehensive model for Mrc1 is one in which Mrc1 is involved in molecular interactions thatcoordinate DNA synthesis and unwinding, i.e., polymerase and helicase, during normal DNAreplication as well as during the checkpoint (Szyjka et al., 2005; Tourriere et al., 2005). In thiscapacity it could promote fork progression but would also be poised to inhibit fork progressionduring replication stress and stabilize the stalled fork.

In this work we present evidence that Mrc1 interacts not only with Cdc45 and the MCMs, butalso with pol ε the primary leading strand replicative polymerase (Morrison et al., 1990; Pursell

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et al., 2007; Shcherbakova and Pavlov, 1996), which is also implicated in checkpoint signaling(Navas et al., 1996; Navas et al., 1995). This dual interaction supports the helicase/polymerasecoupling function for Mrc1.

RESULTSYeast Two Hybrid Experiments Reveal a multi-site Pol2/Mrc1 Interaction

Pol ε consists of four subunits, Pol2, Dpb2, Dpb3, and Dpb4 (Kawasaki and Sugino, 2001 forreview). Pol2 is the catalytic subunit (Fig. 1A). The Pol2 N-terminal half (Pol2N, residues 1–1264) encodes the polymerase and proofreading nuclease active sites. Interestingly, theremaining, noncatalytic 120 kDa Pol2 C terminus (Pol2C, residues1265–2222), comprises theonly essential part of the protein. A mutant lacking Pol2N, pol2-16, is viable (Dua et al.,1999; Kesti et al., 1999). We carried out a two-hybrid screen to identify proteins that interactwith Pol2C in hopes of defining its essential functions (Edwards et al., 2003). One of the genesidentified, YCL061C, encodes residues 251–983 of MRC1, and full-length Mrc1 was alsofound to interact with Pol2C (Fig. 1B). Among other Pol2-interacting proteins (Dua et al.,2000; Edwards et al., 2003), Dpb2 appears to interact weakly with Mrc1, but DPB3, DPB4,and DPB11 do not (Fig. 1B). [Further mapping of the interaction domain within Pol2C is shownin Figure S1.] These results suggest that Mrc1 may interact directly with Pol2 and that Dpb2may modulate that interaction.

Meanwhile, we were surprised to find that Mrc1 interacts with Pol2N, in addition to Pol2C(Fig. 1B). Thus, there are two independent interaction sites for Mrc1 in Pol2.

We also found that mrc1AQ binds robustly to Pol2C (Fig. 1B). Since Mec1-dependentphosphorylation of Mrc1 does not appear to be essential for interaction, we conclude that Mrc1may interact with Pol2 in normal replisome.

Pol2/Mrc1 Physical InteractionTo verify that the two hybrid results reflected physical interactions between the endogenousproteins, we studied the Mrc1/Pol2 interaction by coimmunoprecipitation. To facilitatedetection of Mrc1 and Pol2 without altering the normal timing of expression or endogenouslevels of the proteins, immunoprecipitation was carried out by tagging chromosomal MRC1with 13 myc epitopes and POL2 with 3 HA (hemagglutinin) epitopes. Both tagged genes arefunctional (Alcasabas et al., 2001; Aparicio et al., 1997; Edwards et al., 2003). As shown inFigure 2A, lanes 1–6, untagged Mrc1 or Pol2 controls are not precipitated by anti-myc or anti-HA antibody. Tagged Mrc1-13myc and Pol2-3HA do coimmunoprecipitate, however,regardless of which species is immunoprecipitated (Fig. 2A, lanes 7–12). Comparison of theamount of protein found in the whole cell extract and immunoprecipitation supernatant,suggests that about half of the cellular Mrc1 associates with Pol2, while almost all Pol2molecules bind to Mrc1.

The interaction is strong enough to be observed in the absence of crosslinking and is stable atphysiological salt concentrations (Fig. 2B). It is also resistant to DNaseI, and therefore isprobably mediated by protein/protein and not protein/DNA interaction.

Because the cells used for these experiments were not synchronized, the fact that all of the Pol2coimmunoprecipitated with Mrc1 suggested that Pol2 may bind to Mrc1 throughout the cellcycle. To investigate this we used cells arrested at G1 phase with α factor or cells released fromα factor and allowed to progress into S phase for 60 min, as determined by flow cytometry.Pol2 and Mrc1 interacted in both G1 and S phase cells (Fig. 2C). Pol2(Dpb2) and Dpb11, amajor participant in replication fork assembly and in the S phase checkpoint and Pol2(Dpb2)

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and GINS also interact in both the presence and absence of DNA synthesis (Masumoto et al.,2000).

To confirm the two hybrid assay result showing that Pol2N and Pol2C interacted independentlywith Mrc1 by coimmunoprecipitation, we first used a competition binding protocol. Weoverexpressed untagged Pol2N or Pol2C in an Mrc1-13myc, Pol2-3HA-containing strain.Pol2N and Pol2C each competed with Pol2, reducing the amount of Pol2 thatcoimmunoprecipitates with Mrc1 and increasing the amount of free Pol2 (Fig. 3A, comparelanes 1–6 with lanes 7–12). Thus, Pol2N and Pol2C can both interact with Mrc1 (see also Fig.1B, 3D, 5, and Fig. S1B).

Mrc1 contains two independent Pol2 interaction domainsThe interaction of Pol2N and Pol2C with Mrc1 suggested that there might also be two Pol2interaction sites within Mrc1. To test this prediction, a set of C-terminal and N-terminaldeletions of MRC1 was generated (Fig. 3B). The deletion mutants are referred to herein by thenumbers corresponding to the amino acids contained in the fragments expressed in the mutants.The MRC1 gene was precisely replaced with either C-terminal, FLAG-tagged Mrc1 fragmentsor N-terminal, HA-tagged Mrc1 fragments. As shown by flow cytometry (Fig. 3C), both N-terminal and C-terminal deletions cause a delay in completion of S phase. mrc1(1–219), mrc1(1–418) and mrc1(1–653) profiles resemble the slow S phase of the mrc1Δ. N-terminaldeletions mrc1(312–1096), (567–1096), and (752–1096) all show a delay in entry into S phaseand slow appearance of 2C DNA content. The HU sensitivity phenotypes of the mrc1 mutantsare shown in Figure S2. Complete characterization of these mutants will be reported elsewhere(Komata and Shirahige, unpublished results).

The POL2 gene in each mrc1-FLAG variant was then tagged with 3HA, and the POL2 genein each mrc1-HA variant was tagged with 13myc. Mrc1 was immunoprecipitated with eitherFLAG or HA antibody. The deletion mutant proteins are expressed and immunoprecipitatedat equivalent levels (Fig. 3B). The anti-Pol2 Western blots of the immunoprecipitates showthat Pol2 coimmunoprecipitates with all N-terminal Mrc1 fragments, although 1–219 interactsmore weakly than the others (lanes 1–5). In addition, Pol2 also coimmunoprecipitates withMrc1 C-terminal fragments, although 567–1096 shows reduced affinity. Binding of full-lengthPol2 to both mrc1(1–418) and mrc1(567–1096), which don’t overlap (Fig. 3B, lanes 4 and 9,red), and to mrc1(1–219) and mrc1(318–1096) which also fail to overlap (Fig. 3B, lanes 5 and8, blue), is consistent with there being an extended zone of Pol2 binding.

N-terminal Sequences in Mrc1 Interact with Pol2N and C-terminal Mrc1 Sequences Interactwith Pol2C

To test the extended interaction domain in Mrc1, we first used a competition-binding assay.As shown in Figure 3B, lane 5, full length Pol2 interacts with mrc1(1–219). We found thatexpression of excess untagged Pol2N or excess full-length untagged Pol2 (Fig. S3, lanes 1–3,lanes 7–9), but not excess untagged Pol2C (lanes 4–6) competes with Pol2-myc for binding tothe N-terminal mrc1(1–219)-HA fragment. Thus, Pol2N binds mrc1(1–219) and Pol2C doesnot.

We then showed by direct immunoprecipitation that HA-Pol2N binds mrc1(312–1096)-FLAGbut not to mrc1(567–1096)-FLAG (Fig. 3D, rows 1–5). Thus, C terminal sequences beyond567 are not required for Pol2N binding. Conversely, HA-Pol2C binds mrc1(567–1096)-FLAGand, barely detectably, even to mrc1(752–1096)-FLAG (Fig. 3D, rows 6 and 7). The strongerinteraction of mrc1(567–1096) with Pol2C found here compared to interaction with full-lengthPol2 in Figure 3B is likely due to the overexpression of HA-Pol2C. [We do not yet understandthe cross-reacting band just below Pol2 that appears only in strains carrying the mutants shown

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in Figure 3D, rows 6 and 7, but Figure 3B shows that even full length Pol2 barely interactswith mrc1(752–1096)]. From these data we can conclude that the Pol2N catalytic domain bindsthe N-terminal, phosphorylation site-rich half of Mrc1 and that Pol2C binds the C-terminalhalf of Mrc1.

The Pol2/Mrc1 Interaction is FunctionalTo obtain evidence that Mrc1 and Pol2 interact functionally, we asked whether Mrc1 wasrequired for the viability of various pol2 mutants or for their survival in the presence of variousDNA synthesis inhibitors. pol2-16, lacking the entire N-terminal nuclease/polymerase domainof Pol2 (residues 176–1134), is both DNA replication and checkpoint proficient, althoughreplication forks move slowly and pol2-16 fails to grow at 37°C (Dua et al., 1999; Kesti et al.,1999; Navas et al., 1995; Ohya et al., 2002). pol2-16 associates with origins of replication inS phase and is as efficient in associating with Dpb2, Dpb3, and Dpb4 subunits as Pol2 itself.Therefore, its essential function is likely in DNA replication. As tabulated in Figure 4A,MRC1 is required for the growth of pol2-16. In contrast, strain pol2-16 mrc1AQ is viable,showing that Mrc1 checkpoint mediator function is not required for pol2-16 viability. Thus,interaction of Mrc1 with Pol2C (1135–2222) may play an important role in DNA replicationor some other essential process that does not require phosphorylation. pol2-16 rad9Δ is alsoviable.

To identify the region of Mrc1 required to support pol2-16 growth, we determined which ofthe mrc1 C- and N-terminal deletion mutants was viable in combination with pol2-16. Theresults in Figure 4A show that mrc1(1–843) pol2-16 double mutants are viable but mrc1(1–218) pol2-16, mrc1(1–418) pol2-16, and mrc1(1–655) pol2-16 are inviable, suggesting thatthe region of Mrc1 between 655 and 843 is important for the replication function of pol2-16.Supporting this interpretation, a double mutant containing both the pol2-16 allele and mrc1(567–1096), which does interact with Pol2C, is viable, but pol2-16 mrc1(752-1096), whichbarely interacts with Pol2C (Fig. 3D) is inviable. Thus, the ability of Mrc1C to interact stronglywith Pol2C correlates with the ability to support Pol2C-mediated roles in DNA replication inpol2-16. Therefore the Mrc1C interactions reported in Figure 3 may be functional at thereplication fork in vivo.

We also tested whether the Pol2 C-terminal point mutant pol2-11 and the pol2 partial deletionmutants shown in Figure 1A required Mrc1 for viability (Fig. 4 and S4). Mutant pol2-11 carriesa stop codon at residue 2196 and, unlike pol2-16, is both replication and checkpoint deficient,similar to mrc1 null mutants (Budd and Campbell, 1993; Dua et al., 1998; Frei and Gasser,2000; Navas et al., 1995). The pol2-11 mrc1AQ double mutant is no more sensitive to the Sphase checkpoint inducers HU and MMS (methyl methane sulfonate) than pol2-11 (Fig. S4).This suggests that mrc1AQ and pol2-11 are epistatic for the S phase checkpoint. However,pol2-11 mrc1Δ was found to be inviable, suggesting but not proving that Mrc1 is importantfor replication in the pol2-11 mutant (Fig. 4A).

Deletion of Pol2C is lethal, and therefore the role of the Mrc1N/Pol2N interaction inmaintaining cell viability could not be tested in the absence of Pol2C. However, pol2-18, atemperature-sensitive mutant that retains high viability in HU, has a mutation in the N-terminalcatalytic domain, Pro710Ser (Araki et al., 1992; Navas et al., 1995). The roles of Pol2C andPol2N in vivo are partially independent, since pol2-18 and pol2-11 show partial interalleliccomplementation, as do pol2-18 and pol2-F, another C-terminal mutant (Dua et al., 1998;Navas et al., 1995). pol2-18 mrc1Δ is inviable, suggesting that Mrc1N/Pol2N interaction maybe functional (Fig. 4A). The poor growth and lethality of pol2-18 mrc1(567-1096) and pol2-18mrc1(752-1096) support this idea. pol2-18 mrc1AQ is viable, suggesting phosphorylation isnot required to correct the pol2 defect, but that Mrc1 is providing some other function.

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We also tested whether plasmids overexpressing MRC1 could suppress the replication and/orreplication stress response defects, including HU and MMS sensitivity, of the pol2-16 andpol2-11 mutants. As shown in Figure 4B and 4C, respectively, overexpressing MRC1ameliorates the temperature sensitivity and DNA damage sensitivity of pol2-11 and bothMRC1 and mrc1AQ suppress pol2-16 growth and weak HU sensitivity. These studies show afunctional in vivo interaction between Mrc1 and Pol2, most likely in DNA replication, sinceMrc1 phosphorylation is dispensable.

Role of the Mrc1/Pol2 Interaction in the S phase Checkpoint: Effect of S/TQ Phosphorylationon Mrc1

Having provided evidence that Mrc1 and Pol2 interact during DNA replication (strictlyspeaking when Mrc1 is not phosphorylated) we wished to determine if their interaction playedany role in the S phase checkpoint. Two hybrid assays showed that mrc1AQ interacted robustlywith POL2C (Fig. 1B). Mrc1AQ was also found to coimmunoprecipitate efficiently with Pol2(Fig. 5A), confirming that phosphorylation is not essential for the Pol2/Mrc1 interaction.

This raised the question of whether phosphorylated Mrc1 binds to Pol2 after checkpointactivation. Mrc1 becomes hyperphosphorylated in cells treated with HU, and thisphosphorylation results in a Mec1/Ddc2-dependent reduced mobility on SDS gels (Fig. 5B,lanes 1–3, note Mrc1 doublet) (Osborn and Elledge, 2003). Clearly, both phosphorylated andunphosphorylated Mrc1 coimmunoprecipitate with Pol2. The non-phosphorylatableMrc1AQ also associates with Pol2 in the presence of HU (Fig. 5B, lanes 4–6). Sincephosphorylated Mrc1 can interact with Pol2, this suggests that Pol2 and Mrc1 continue tointeract physically during the checkpoint cascade.

Phosphorylation of Mrc1 Abolishes Interaction with Pol2N but not with Pol2C, Suggesting aRole for Pol2N in Checkpoint Responses

Although phosphorylation of Mrc1 did not reduce binding of Mrc1 to full-length Pol2, wewished to determine if phosphorylation of Mrc1 might affect binding to either of the individualPol2 halves. When either HA-tagged Pol2N or HA-tagged Pol2C was expressed from a highcopy plasmid, pACT2, under the control of the strong ADH promoter, the respective taggedPol2 domain coimmunoprecipitated with endogenous Mrc1-13myc (Fig. 5C, row 1, lanes 6and 12), confirming that Pol2N and Pol2C interact independently with Mrc1. (The untagged,chromosomal POL2 gene is present to preserve viability.) Mrc1 in extracts of cells treated with150 mM HU migrated more slowly than Mrc1 in untreated cells, presumably due tophosphorylation (Fig. 5C, row 1, and compare lanes 1–6 with 7, 8 or lanes 10–12 with 13–15).Strikingly, HA-Pol2N fails completely to interact with Mrc1-P (Fig. 5C, row 1, lane 9, androw 2, long exposure), while HA-Pol2C binds Mrc1-P avidly (Pol2C IP, row 1, lanes 13–15;Mrc1 IP, rows 8 and 9, lanes 13–15).

By reducing the levels of HU to either 100 mM HU or to 75 mM HU, we could study theinteraction where only a fraction of the Mrc1 was phosphorylated, and both Mrc1 and Mrc1-P were present in the same cells. We showed clearly that HA-Pol2N binds non-phosphorylatedMrc1 (faster migrating) but not Mrc1-P, which remains in the supernatant (Fig. 5C, row 4 androw 6, lanes 7–9). Conversely, both Mrc1 and Mrc1-P species bind to HA-Pol2C in these cells(Fig. 5C, row 4 and row 6, lanes 13–15).

Therefore, phosphorylation of Mrc1 affects binding to the N- and C-terminal halves of Pol2differentially. Mrc1-P is released from the Pol2N “catalytic” domain but remains bound to thePol2C “structural” domain. These results suggest a role for the Mrc1N-Pol2N interaction inthe checkpoint pathway.

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Deletion of Mrc1 destabilizes Pol2 at replication forks stalled in the presence of HUTo test the idea that Mrc1 affects Pol2 at replication forks during the checkpoint, we carriedout chromatin immunoprecipitation experiments using an oligonucleotide array capable ofanalyzing protein binding to yeast chromosome VI at 600 bp resolution (Katou et al., 2003).As shown in Fig. 6A, Pol2 is stable at replication forks after 60 minutes of treatment with HUin wild-type cells. In an mrc1Δ, however, Pol2 is clearly destabilized. It has been previouslydemonstrated that Tof1, which is found in a complex with Mrc1 and Csm3, is required forloading of Mrc1 onto chromatin (Katou et al., 2003). If the destabilization of Pol2 in themrc1Δ was due to lack of Mrc1 at the replication fork, then we expected that Pol2 should alsobe destabilized in a tof1Δ. As shown in Fig. 6A, this is the case. We conclude that the Pol2interaction at the fork is destabilized in the absence of Mrc1. Pol2 may have spread out on thechromatin such that it is undetectable or may have dissociated altogether; however, unlike otherreplication proteins previously tested (Katou et al., 2003), it is not associated with thereplication fork.

Coimmunoprecipitation of Mrc1, Pol2, and Mcm2Mrc1 coimmunoprecipitates with Cdc45 and copurifies with MCM-CDC45 (Gambus et al.,2006; Katou et al., 2003), and it has been proposed that Mrc1 couples unwinding by the MCMhelicase and DNA synthesis (Szyjka et al., 2005). Our results are compatible with coupling, atleast on the leading strand, occurring through Pol2, so we asked whether both Mrc1 and Pol2coimmunoprecipitated with Mcm2. We combined Mcm2-13myc, Pol2-3HA, andMrc1-6FLAG in the same strain, all expressed from their native promoters, and carried out aMcm2-myc immunoprecipitate with anti-myc antibody (Fig. 6B). Both Mrc1 and Pol2 wererecovered in the Mcm2-immunoprecipitate. This result makes it possible that all three proteinsinteract simultaneously, although the experimental design does not distinguish between theexistence of a single complex with all three proteins and two different Mcm2/Mrc1 and Mcm2/Pol2 complexes. We then carried out the same experiments in HU-treated cells. Mcm2coimmunoprecipitates with both phosphorylated and unphosphorylated Mrc1. The interactionbetween Mcm2 and Mrc1-P is an important and novel result showing that Mrc1 interacts withthe MCM complex not only during the normal cell cycle but also during the checkpoint, as itdoes with Pol2 (Fig. 3 and 5).

DISCUSSIONOur results provide a new perspective on Mrc1, on pol ε and on sensing and transmission ofsignals in the S phase checkpoint. Our results imply at the molecular level how Mrc1 couldmediate by a common mechanism, i.e., coupling between helicase and polymerase, its twoapparently disparate functions, namely stimulation of replication during S phase and inhibitionof replication during stress.

The Role of Mrc1 in DNA ReplicationWe have shown that all of the Pol2 in the cell is associated with Mrc1. This stoichiometry alonesuggests that Mrc1 is very important for all of the roles played by pol ε. The suppression ofpol2-11 and pol2-16 phenotypes by overexpression of Mrc1 suggests that Mrc1 either stabilizespol ε directly or a complex containing pol ε. Since Mrc1 is not essential, it cannot be essentialfor wild-type pol ε at replication forks. We showed, however, that interaction between Mrc1Cand Pol2C is likely important for the essential function of Pol2 and for the replication functionof Mrc1, since non-phosphorylatable Mrc1 can support the viability of a number of differentC-terminal pol2 mutants. Cryoelectron microscope reconstruction of the pol ε holoenzymesuggests that Pol2C sits as a tail on the globular Pol2N domain (Asturias et al., 2006 andreferences therein). Thus, Pol2C likely exerts its essential but non-catalytic role as a scaffoldfor Dpb2, Dpb3, and Dpb4, which together appear to be involved in guiding the primer/template

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into the Pol2N active site (Fig. 7A). The essential function for the Pol2 C-terminal scaffold inpol2-16 mutants suggests that Pol2C is also a mainstay of the entire replisome (Dua et al.,1999). Mrc1 might therefore enhance the function of the Pol2 C-terminal domain inmaintaining a stable replisome.

In addition to binding the C-terminal half of Pol2, the interaction of Mrc1 and Pol2 extendsover the entire protein. Mrc1 also interacts with the polymerase/exonuclease domain,suggesting that Mrc1 may also serve as an accessory factor in polymerization by Pol2. It hasbeen proposed that Mrc1 binds to DNA, and Mrc1 might affect the association of pol ε withthe primer terminus. Our flow cytometric analysis clearly shows that deletions within theMrc1N with Pol2 do have S phase defects. Additionally, Mrc1N might help recruit, make theprimer accessible to, or stabilize another DNA polymerase, for instance one that compensatesfor the absence of the Pol2 polymerase in pol2-16, such as pol δ, or that participates in DNAdamage repair or by-pass. pol2-16 is synthetically lethal with cdc2-1, the catalytic subunit ofpol δ pol32Δ, a non-essential subunit of pol δ, and pol3-01, a mutant defective in theexonuclease proofreading domain of pol δ. A reservation in deducing that Pol2/Mrc1interaction functions during normal DNA replication is that we have only shown that theinteraction occurs in S phase and not that it occurs on chromatin. Since all of the Pol2coimmunoprecipitates with Mrc1 in S phase, however, at least some interaction likely occurson the DNA.

Polymerase/Helicase CouplingCoimmunoprecipitation between Mrc1, Pol2, and MCM2 (Fig. 6) during a normal S phase andbetween Mrc1 and MCM2 during HU treatment, i.e., during the checkpoint, are consistent withthe proposal that Mrc1 might enhance interaction between Pol2 and the replication helicase.A fraction of the cellular pol ε pool has also been shown by others to coimmunoprecipitatewith Cdc45, and this is specific to pol ε since pol α does not associate with Cdc45 (Zou andStillman, 2000). These results provide molecular evidence for the proposal that Mrc1 may beinvolved in coupling polymerase and helicase function, originally proposed based on observedfunctional uncoupling of DNA synthesis and unwinding in mrc1 null strains in the presenceof replication stress (Szyjka et al., 2005). GINS, a third component of the RPC, is almostcertainly also involved in coupling, and the association of Mrc1 with the RPC also indirectlysupports a role for Mrc1 in coupling. A model for Mrc1/Pol2/MCM coupling is shown in Figure7A. A parallel coupling model has been proposed for the lagging strand in which Mcm10 andAnd-1/Ctf4 have been proposed to link pol α and the MCM helicase (Zhu et al., 2007).

At the prokaryotic replication fork, polymerase/helicase coupling is both required formaximum elongation rates and also appears to be at the root of a replisome in which thepolymerases (are programmed to?) transiently dissociate from the primer/template and yetremain associated with the moving fork through interaction with the other proteins at the fork.This “dynamic processivity” is accomplished by direct interaction of the polymerase with thehelicase in phage T7, indirect polymerase/helicase interaction mediated through the clamp-loader component of the pol III holoenzyme in E. coli, and possibly by interaction with theclamp subunits in phage T4. It is easy to envision how this transient mode of interaction of thepolymerases with the primer terminus but processive interaction with the fork provides forrelease and rapid recycling of polymerases to new primers on the lagging strand as well as forrapid exchange to “reserve” polymerases when damage to the template or to the engagedpolymerase or barriers in the template are encountered. This also provides a new way to viewthe coordination of leading and lagging strands (Lovett, 2007). Indirect evidence for replicativeDNA polymerase interchangeability in eukaryotes (between an actively synthesizingpolymerase and a “reserve” polymerase) is provided by the pol2-16 mutant, in which a second

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polymerase, presumably pol δ, can fulfill the role of the missing polymerase of pol ε on theleading strand (Dua et al., 1999).

S Phase Checkpoint Function of the Mrc1/Pol2 InteractionMrc1 was first described as a mediator between the signaling and effector kinases, Mec1 andRad53, and as such regulates the global checkpoint responses, such as slowing of the cell cycle,induction of transcription, and inhibition of late origin firing. Our results suggest Mrc1 mightalso be involved in sensing and additional signaling mechanisms. Association with Pol2Nplaces Mrc1N near the polymerase active site, where it might sense replication fork blocksgenerating a checkpoint signal (Fig. 7B). When Mrc1N is phosphorylated, presumably byMec1, in response to replication stress (HU addition), Mrc1N dissociates from Pol2N (Fig. 5).One possible consequence of this dissociation might be slowing of the replication fork. In thiscase, Mrc1 would itself be an effector of the S phase checkpoint. Release could also allowMrc1-P to interact with another protein, perhaps an MCM component or Cdc45, at thereplication fork, inhibiting fork movement.

Another consequence of the release of the phosphorylated Mrc1 N terminus might berecruitment, stabilization, and/or activation of Rad53, in turn activating the downstreamcheckpoint events. Alternatively, the release of Mrc1N might cause a conformational changein Pol2C that leads to activation of the checkpoint through the C-terminal checkpoint domain.If so, then Pol2N may constitute the elusive suppressor of checkpoint activation postulated tooperate during normal, unimpeded DNA replication. In sum, the results suggest a molecularmechanism by which DNA damage on the leading strand can be sensed and the signal amplifiedthrough activation of downstream kinases.

Concomitant with sensing and signaling through its bimodal N-terminal interactions withPol2N, Mrc1 might be involved in stabilizing the fork through its continued interaction withPol2C during the checkpoint, in preparation for replication restart. Stabilization of thereplication forks is often considered the most important function of Mec1 (Pasero et al.,2003 for review). Mrc1C might be acting to anchor Pol2C to the helicase and to the primer/template junction during the checkpoint. In the complete absence of Mrc1, both Pol2N andPol2C may have weakened interaction with primer/template. Unwinding can still occur butsynthesis ceases (Nedelcheva et al., 2005).

A critical finding is that Pol2, which is associated with HU-stalled replication forks in wild-type cells, is destabilized at stalled forks in mutants lacking Mrc1 or in mutants where Mrc1fails to associate with DNA (Fig. 6A). In this sense Pol2 is exceptional, since in previous studiesmost replication proteins tested were shown to be uncoupled from the site of DNA synthesisbut remained colocalized on the chromatin at a distal site (Katou et al., 2003). Unlike Pol2,Dpb3, a subunit of pol ε, remains with the replisome in the mrc1 Δ mutant in the presence ofHU (Katou et al., 2003). This discrepancy could be explained by the fact that Dpb3 and Dpb4,histone fold-containing proteins encoded by non-essential genes (Iida and Araki, 2004), forma subcomplex independent of Pol2/Dpb2 (Dua et al., 2000;Dua et al., 2002). The Dpb3/Dpb4complex may be stabilized by interactions with fork components or chromatin componentsother than Mrc1. Dpb3 and Dpb4 may also be components of chromatin remodeling complexes(Iida and Araki, 2004). Pol2 association with ARS607 is also significantly reduced in mrc1Δcompared to wild-type after 60 minutes of HU treatment in an independent study (Bjergbaeket al., 2005).

The Pol2/Mrc1 interactions described here may be conserved in metazoans. Metazoan Claspinis a functional Mrc1 ortholog. An evolutionary comparison of Mrc1/Claspin structure, as wellas a summary of the interaction data presented in this paper, are shown in Figure S5,emphasizing that although the functions are conserved, the overall organization of the proteins

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differ. Similar to our observations in yeast, pol ε is associated with Claspin immunoprecipitatedfrom aphidicolin-treated Xenopus egg extract chromatin eluates (Lee et al., 2005). Furthermore,hyperunwinding that involves uncoupling of the polymerase and the helicase has been observedin Xenopus in the presence of aphidicolin, a polymerase inhibitor (Byun et al., 2005; Walterand Newport, 2000).

EXPERIMENTAL PROCEDURESYeast Strains and Plasmids

Strains, plasmids, and oligonucleotides used in this study are listed in Tables S1-S3.

Analysis of Double MutantsViability of double mutants carrying various MRC1 alleles and either pol2-11, pol2-16, orpol2-18 alleles was determined by two methods. First, spores from standard crosses and tetraddissection were incubated and genotyped at 23°C. Any combination failing to generate viabledouble mutants was deemed synthetically lethal. At least 12 tetrads were dissected for eachdouble mutant, and usually more were dissected. Second, POL2/pol2 and MRC1/mrc1heterozygous diploids carrying plasmid encoded MRC1-URA3 (cloned in pRS416) weregenerated for each pol2 and mrc1 mutant. mrc1 pol2 double mutants were identified after tetraddissection and then tested for viability in the absence of MRC1-URA3 by plating on 5-FOA.

Construction of Mrc1 Deletion MutantsPCR products of the Mrc1 promoter region (300 bp upstream of the MRC1 gene) were obtainedusing specific primers, as indicated in Table S3. The PCR product was digested with BglII andPacI and ligated into BglII/PacI digested pFA6a-His3MX6-PGAL thus replacing PGAL1 withthe Mrc1 promoter. Mrc1 N-terminal deletion mutants were generated using this plasmid andMrc1 FLAG strain which was deleted 200 bp upstream of the MRC1 gene with URA3 marker.Mrc1 C terminal deletions were constructed using the primers indicated in the Mrc1-HA strain.

Cell SynchronizationStrains were arrested in G1 phase by supplementing the media of exponential cultures with 10μg/ml αfactor. Synchronized cells were released from G1 into fresh YPD and growth continuedfor 60 min, after which all cells showed S phase DNA content by flow cytometry (Reis andCampbell, 2007).

Immunoprecipitation AssaysCoimmunoprecipitation analysis of wild-type or mutant Mrc1 with Pol2 was performed usingstrains coexpressing a 13myc-tagged version of Mrc1 and 3HA tagged version of Pol2. Othertags employed for specific experiments are described in the legends to the figures. Samples(2.0×109 cells) were collected by centrifugation and the extract was prepared as previouslydescribed. The cells were crushed by glass beads in 180 μl lysis buffer [45 mM Hepes-KOHpH 7.2, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% NP40] containing protease inhibitors[1% Protease Inhibitor Cocktail (Sigma) and 1 mM PMSF] and cleared by centrifugation(WCE). WCE total protein (6 mg) was incubated with 6 μg of antibody indicated in the figuresfor 3h at 4°C. anti-myc antibody was monoclonal 9E10 and anti-HA antibody was 12CA5.anti-FLAG antibody was from Sigma Aldrich. Precipitation was carried out with pre-washedprotein G beads (GE Healthcare) for 3h at 4°C. The supernatant was recovered and washed 4xwith lysis buffer (or with increasing NaCl concentrations where indicated). To degradechromosomal DNA in cell extracts, DNase I (Worthington) was added to a final concentrationof 200 units per ml and incubated for 1–2h at 4 °C before IP. DNA degradation was confirmedby agarose gel electrophoresis and EtBr staining.

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Antibody-bound fractions were suspended in a minimal amount of SDS loading buffer (62.5mM Tris–HCl pH 6.8, 2% sodium lauryl sulfate, 10% glycerol, 5% 2-mercaptethanol) and halfof the sample was analyzed by Western blotting using an ECL detection reagent (Amersham).WCE and SN (immunoprecipitate supernatant), both corresponding to approximately 100 μgtotal protein, were also analyzed.

Competition binding assays were carried out using the same strains as standardcoimmunoprecipitations, except that strains contained plasmids (pBTM116) overexpressinguntagged Pol2, Pol2N, or Pol2C. To determine binding of Pol2N and Pol2C to Mrc1 directly,Mrc1 tagged strains were transformed with pACT2 plasmids overexpressing HA-tagged Pol2,Pol2N, and Pol2C. Pol2C (residues 1265–2222) carrying a series of 10 amino acid deletionsin the C-terminal 120 amino acids as well as the pol2-11 mutant were cloned into pACT2,which overexpresses HA tagged versions of the mutant proteins (Dua et al., 2000; Dua et al.,1999; Dua et al., 1998). These constructs were introduced into an Mrc1-13myc strain. We thencarried out myc IPs to detect interaction between Mrc1 and pol2-HA mutant proteins.

(ChIP)-chip AssaysThe oligonucleotide arrays of chromosome VI with 300 nucleotide resolution, chromatinimmunoprecipitation procedures, internal controls, and strains used have been described(Katou et al., 2003). Strains contained Pol2-3HA.

Data in this paper can be obtained from GEO (http://www.ncbi.nlm.nih.gov/geo) withaccession number GSE12138.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Stephen Elledge and Oscar Aparicio for strains. This work is dedicated to a gifted teacher, Ernest Russ. Thiswork was supported by MEXT Japan, Grant-in-Aid for Scientific Research on Priority Areas, “Chromosome Cycle”to K.S. and M.K., GCOE program from MEXT Japan to Y.K., a CIRM fellowship to HL and NIH GM25508 and NIHGM087666 to JLC.

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Figure 1. Mrc1 Interacts with Pol2(A) Schematic map of the full-length POL2 gene and the zinc finger region. The full-lengthgene, 2222 residues, is shown at the top. Pol2N: Five conserved nuclease motifs, residues 288and 501; seven partially conserved DNA polymerase motifs, residues 544 and 954, threeconserved motifs of unknown function, residue 954 and 1183 (not indicated here); Pol2C:essential “spacer” region conserved only in the Pol2 subfamily of B family polymerases, 1180and 2103; conserved zinc finger motif, 2103–2222(end). MutA through MutI are deletionswithin the zinc finger domain used for mapping the interaction subdomain in Figure S1 (Duaet al., 1998).(B) Two hybrid assay for Mrc1/Pol2 interaction. Analyses were performed on at least threeindependent transformants as described previously (Edwards et al., 2003).

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Figure 2. Reciprocal Coimmunoprecipitation of Pol2 and Mrc1Mrc1-13myc/Pol2-3HA (strain AC2301, Table S1) is the doubly tagged strain used to testcoimmunoprecipitation. Whole cell extracts (WCE) were prepared from asynchronous cells asdescribed in Experimental Procedures. Immunoprecipitation (IP) was carried out with theantibody indicated, and proteins in the WCE, the supernatant (SN), and the IP were determinedby Western blotting. WB=Western Blot. WCE and SN represent the same number of cellequivalents of protein. In control experiments, 10x excess of SN was also loaded to verifyestimated precipitation efficiency.(A) Coimmunoprecipitation. Lanes 1–6: Mrc1-13myc/Pol2 indicates strain Y1134 carryingmyc-tagged Mrc1 and untagged Pol2 and serves as a control that anti-HA does notimmunoprecipitate Mrc1 when Pol2 is untagged. Mrc1/Pol2-3HA, strain A1591, serves as acontrol that anti-myc does not immunoprecipitate Pol2-HA when Mrc1 is untagged. Lanes 7–12: As indicated in the figure, Pol2-3HA was precipitated with anti-HA or Mrc1-13myc wasprecipitated with anti-myc, and the amount of Pol2 and Mrc1 in the respectiveimmunoprecipitate was determined as described in Experimental Procedures.(B) Salt and DNase I sensitivity of coimmunoprecipitation. Lanes 1–8, wash buffers containedthe amounts of NaCl indicated. Lanes 9–12. Extracts were treated with DNase I before IP.Immunoprecipitation was carried out as in A.(C) Pol2/Mrc1 interaction as a function of cell cycle stage. Tagged strains indicated at the topof the figure were staged in the cell cycle as described in Experimental Procedures.Asynchronous cells (Asyn), cells arrested with α factor (G1), cells released from α factor-arrestinto S phase (S). Immunoprecipitations were carried out with the indicated antibodies. Westernblots of the precipitates are shown.

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Figure 3. Two Independent Interactions between Pol2 and Mrc1(A) Competition binding assays show Pol2N and Pol2C bind Mrc1. An anti-myc IP was carriedout on strain AC 2301, MRC1-myc/POL2-HA, carrying plasmid pBTM116 or pBTM116overexpressing either Pol2C or Pol2N as potential competitors for POL2-HA binding toMRC1-myc. Lanes 1–3, no plasmid; lanes 4–6, pBTM116 vector; lanes 7–9, pBTM116expressing Pol2C; lane 10–12, pBTM116 expressing Pol2N. Blots of IPs in were probed withanti-HA antibody in the upper row and on a separate gel with myc antibody in the lower row.(B) Interaction of Mrc1 deletion mutants with Pol2. A schematic diagram of the MRC1 geneand the deletion mutants is shown. P’s represent likely Mec1/Ddc2 phosphorylation sites. Theindicated N-terminal mrc1 deletion mutants were tagged with FLAG epitope and the C-terminal mrc1 deletion mutants were tagged with HA epitopes (see Experimental Proceduresand Table S1). The POL2 gene was tagged with 13myc in strains AC2200-2204 (C-terminalmrc1 deletions) or with 3HA in strains AC2205-2209 (N-terminal mrc1 deletions) byhomologous recombination as indicated. Immunoprecipitation was carried out with anti-FLAGantibody for the N-terminal Mrc1 deletions and anti-HA antibody for the C-terminal Mrc1deletions, and immunoprecipitates were probed with anti-FLAG and anti-HA antibody asindicated. Anti-Pol2-myc or anti-Pol2-HAWestern blots of immunoprecipitates revealefficiency of Pol2 coimmunoprecipitating with the respective mrc1 mutant proteins.(C) S phase progression of various mrc1Δ mutants is delayed. Cells were synchronized in G1phase with α factor and then released into the cell cycle. Samples were taken for flow cytometryanalysis at the indicated times.(D) Mrc1N binds Pol2N and Mrc1C binds Pol2C. Binding studies were carried out as in Figure2. Strains contained the respective, chromosomally-tagged mrc1 mutant, a wild-type, untaggedPOL2 chromosomal gene, and HA-Pol2N or HA-Pol2C expressed from a high copy number,ADH-promoter plasmid, pACT2. The species that were immunoprecipitated are indicated onthe left and coimmunoprecipitating species detected by Western blotting are indicated on theright of each panel. Rows 1 and 2: mrc1(312-1096-FLAG) carrying pACT2-HA-Pol2N; rows3 and 4: mrc1(567-1096-FLAG) carrying pACT2-HA-Pol2C; row 5: mrc1-(752-1096-FLAG)carrying pACT2-HA-Pol2C; row 6: mrc1(312-1096-FLAG) carrying pACT2-HA-Pol2N; row7: mrc1(567-1096-FLAG) carrying pACT2-HA-Pol2N.

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Figure 4. Genetic interactions between POL2 and MRC1(A) Phenotypes of various pol2 mrc1 double mutants. These results were obtained using twocomplementary methods for creating and analyzing double mutants, as described in detail inExperimental Procedures. Strain genotypes are shown in Table S1.(B) Suppression of pol2-11 by MRC1 overexpression. 10-fold serial dilutions of exponentiallygrowing cultures of pol2-11 cells transformed with plasmids expressing MRC1 (pACT2-MRC1, pBTM116-MRC1) or control pol2-11 carrying empty pACT2 or without plasmid wereplated and grown at different temperatures or in the presence of HU or MMS as indicated.(C) Suppression of pol2-16 by MRC1 Overexpression. Plates streaked from a single colony ofstrain pol2-16 transformed with plasmids indicated on the left (Table S2) were grown atdifferent temperatures or in the presence of HU as indicated.

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Figure 5. Regulation of Pol2/Mrc1 Interaction by Phosphorylation of Mrc1(A) Pol2 interacts with Mrc1AQ. Anti-myc IP of strain mrc1AQ-myc/POL2-HA, AC2302. IPswere probed with anti-HA antibody to detect Pol2 as in Figure 2C. The experiment was carriedout simultaneously with that shown in Figure 2C.(B) Pol2 interacts with both nonphosphorylated and phosphorylated Mrc1. Mrc1-13myc andPol2-3HA coimmunoprecipitate in cells treated with HU. Strain AC2301, MRC1-myc/POL2-HA (lanes 1–3) or strain AC2302, mrc1AQ-myc/POL2-HA (lanes 4–6) were synchronizedwith α factor and released into fresh medium containing 75 mM HU for 60 min followed byimmunoprecipitation with anti-HA antibody (see text for conditions compared). Mrc1-P refersto phosphorylated Mrc1.(C) Mrc1-P does not interact with Pol2N but does interact with the Pol2-C. Pol2N and Pol2Cwere each tagged with HA in pACT2, and expressed in an Mrc1-13myc strain (Y1134) in theabsence or presence of the indicated amounts of HU. HA-Pol2N or HA-Pol2C was IPed andMrc1 or Mrc1-P was detected in the IP with α-myc (rows 1–7). WB HA indicates the controlfor Pol2N and Pol2C IP efficiencies; rows 3, 5, and 7. Rows 8 and 9 are Mrc1-myc IPs. Row9 is control for Mrc1 IP. The various rows and lanes are described in the text.

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Figure 6. Mrc1 stabilizes Pol2 at stalled forks and connects it with MCM complex(A) Mrc1 stabilizes Pol2 at the replication fork in cells treated with HU. Wild-type, mrc1Δ ortof1Δ strains were synchronized with α factor and released from pheromone arrest in thepresence of 200 mM HU. After 60 min, chromatin immunoprecipitation analysis was carriedout as described previously. The blue histograms represent Pol2 enrichment. The vertical axisis enrichment on a log2 scale. The horizontal axis is length in kb.(B) Mrc1, Pol2, and the MCMs interact in single complex. Coimmunoprecipitation of Mrc1,Pol2, and Mcm2. Mcm2-13myc/Mrc1-FLAG/Pol2-3HA triple tagged strains were used forcoimmunoprecipitation (strains listed in Table S1). Whole cell extracts (WCE) were preparedfrom asynchronous cells (Asyn), cells arrested with α factor (G1), or cells released from αfactor into S phase in the presence of HU treatment (HU). Immunoprecipitation was carriedout as described in the legend to Figure 3 and Experimental Procedures. Lanes 1–3 show acontrol strain with untagged Mcm2. Lanes 1–3, top row, show that Mcm2 was notimmunoprecipitated by the myc antibody. Lanes 1–3, bottom row, show that the Mrc1-FLAGdid not immunoprecipitate with the myc antibody in the absence of myc-tagged Mcm2. Controlshowing that Pol2-HA does not coimmunoprecipitate with myc antibody is shown in Fig. 2A,lane 6.

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Figure 7. Models of Functional Mrc1/Pol2 Interaction on the Leading Strand(A) Interaction during normal DNA replication. The shape of pol ε depicted is derived fromreference (Asturias et al., 2006). The interactions between Mrc1 and Pol2 described in thisstudy are incorporated into a rudimentary model for polymerase/helicase coupling duringnormal DNA replication (see text).(B) Mrc1/Pol2 interaction on the leading strand during replication stress. Pol ε/Mrc1 sensesthe damage, phosphorylation of Mrc1 releases the N terminus, but there is continued interactionbetween Mrc1-P with the C terminus of Pol2.

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