MCB Accepted Manuscript Posted Online 13 April 2015 Mol. Cell. Biol. doi:10.1128/MCB.01357-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Mcm2-7 is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed

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Modulation of its DNA Gate

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Running title- Mcm2-7 coordinates the DNA replication checkpoint

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Feng-Ling Tsaia,*,†, Sriram Vijayraghavana,*,‡, Joseph Prinzb, Heather K. MacAlpineb, David M. MacAlpineb, and Anthony Schwachaa,#

a

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260

b

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710

* #

†

F.T and S.V contributed equally to this work

Address correspondence to Anthony Schwacha, [email protected] Office: (412) 624-4307

Present address-Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University, Baltimore, MD 21287 ‡

Present address-Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710

Word count for Materials and Methods: 883 Combined word count for Introduction, Results, and Discussion:

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Abstract

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prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is

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incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase

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active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as

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part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1)

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phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective

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effector kinase (Rad53) activation and classic DRC phenotypes. Our prior in vitro analysis has shown

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that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We

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infer that this conformational change is required for its DRC role and propose that it allosterically

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facilitates Rad53 activation to ensure a replication-specific checkpoint response.

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The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to

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Introduction

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S-phase, and if irregularities occur, facilitates numerous cellular responses that promote genome

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stability. In budding yeast, this checkpoint depends upon a phosphorylation cascade initiated by the

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upstream sensor kinase Mec1/ATR (1, 2) which in turn leads to the phosphorylation and activation of

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the Mrc1/Claspin mediator (3, 4) and ultimately the effector kinase Rad53/Chk1 (5, 6). During dNTP

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limitation or other forms of replication stress, DRC activation protects genome stability by Rad53-

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dependent phosphorylation of multiple downstream targets that serve to stabilize nascent replication

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forks, and blocks cell cycle progression, inappropriate recombination (7-9) and the activation of late

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origins until the stress is alleviated (reviewed in (10)). In addition to the DRC, a second related

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pathway that specifically monitors and responds to DNA damage and double strand breaks also

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operates during S phase (DNA damage checkpoint (DDC), reviewed in (11)).

The eukaryotic DNA replication checkpoint (DRC) monitors chromosome duplication during

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How the DRC cascade mechanistically interacts with the core replication machinery is

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incompletely understood. Current evidence indicates that replication plays a passive role in the

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process. DNA lesions or stress cause a physical uncoupling between DNA polymerase and the

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replicative helicase; this in turn results in an aberrant increased level of ssDNA production that leads to

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checkpoint activation (12, 13). (14). Correspondingly, normal replication fork formation is a

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prerequisite for DRC activation (15-18).

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However, strong interactions between DRC components and core replication factors, even in

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the absence of replication stress, suggest that DNA replication in general and MCM replicative

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helicase in particular play broader roles in the DRC. The mediator proteins in the cascade

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(Mrc1/Claspin, Tof1/Timeless, Csm3/Tipin) physically interact with and stabilize both Mcm2-7 and

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DNA polymerase ε (19-23) and protect fork integrity during replication stress (21, 24). Moreover,

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these associations are necessary for checkpoint function: loss of physical interaction between Mrc1 and

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the Mcm6 subunit (25) causes DNA damage sensitivity, consistent with a DRC defect. Similarly,

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physical interaction between Mcm7 and Rad17, a component of the checkpoint clamp loader complex

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(Rad17/Rfc2-5) that, together with the 9-1-1 complex, senses replication stress, is required for normal

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DRC activity (26).

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The present study further explores the possible roles of Mcm2-7 in DRC checkpoint activation

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and signal transduction. Mcm2-7 is a toroidal AAA+ ATPase that comprises the catalytic core of the

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replicative helicase that unwinds duplex DNA during replication (reviewed in (27)). The loading and

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activation of Mcm2-7 are key landmark events that ensures a single round of DNA replication occurs

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during each cell cycle (reviewed in (28)). Interestingly, unlike other hexameric helicases, Mcm2-7 has

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a unique heterohexameric subunit composition (Mcm2 through 7) that results in 6 distinct ATPase

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active sites formed at dimer interfaces. This subunit organization allows a division of labor among

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active sites, with several sites dedicated to DNA unwinding, while other sites appear to form and

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possibly regulate a structural discontinuity (the Mcm2/5 gate) within the Mcm2-7 ring structure

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(reviewed in (27).

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The Mcm gate appears to regulate several aspects of Mcm2-7 function. Biochemical evidence

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indicates that the gate-open Mcm2-7 conformation lacks helicase activity while the gate-closed form

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retains activity (29). In vivo, the Mcm gate has two known functions: 1) to serve as an entry site for

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DNA loading during origin association (29) and, 2) to regulate DNA unwinding during G1/S.

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Interestingly, structural evidence shows that Mcm gate closure and subsequent helicase activation

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during the G1/S transition requires the loading of the replication factors Cdc45 and GINS to generate

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the CMG (Cdc45-Mcm2-7-GINS) complex (30, 31). Although the regulation of Mcm gate

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conformation is poorly understood, current information indicates that it is modulated by ATPase active

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sites that flank the gate (i.e., Mcm5/3 and Mcm6/2): mutations in conserved ATPase motifs at these

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sites biochemically bias the Mcm2-7 complex into a gate-closed form (32). We were therefore

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interested in the specific possibility that MCM-DNA gate regulation might come into play during the

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DRC checkpoint response to replication stress.

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To address this, we studied a biochemically characterized viable non-null allele —

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mcm2DENQ — that surgically inactivates the Walker B ATPase motif of the Saccharomyces

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cerevisiae Mcm2, thereby blocking ATP hydrolysis at the Mcm6/2 active site and biasing the ring into

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a gate-closed conformation (32, 33). Our interest was piqued, in part, by the fact that the Mcm6

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subunit has been previously shown to functionally and physically interact with the Mrc1 mediator

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protein (25).

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We characterize below the effects of mcm2DENQ on the DRC and DDC responses. These

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results reveal that Mcm2-7, and specifically the ATPase site inactivated by mcm2DENQ, are required

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at an intermediate step of the DRC signal transduction cascade. We suggest that involvement of

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Mcm2-7 at this step helps to ensure specific discrimination of replication stress from DNA damage

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stress. We propose specifically that this role is conferred directly because the open-gate conformation

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of Mcm2-7 allosterically assists the recruitment of Rad53 to Mrc1 to enable effector kinase activation.

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Materials and Methods

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Yeast Methods. S. cerevisiae strains and plasmids are listed (Supporting Tables 1-2). All strains are

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isogenic derivatives of W303 and construction details are available upon request. For over-expression

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experiments, plasmids containing either MCM2 (pUP221) or mcm2DENQ (pUP223) expressed under

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the galactose-inducible GAL1 promoter were integrated into the LEU2 gene of the indicated strains.

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Cultures were grown at 30°C unless otherwise noted. G1 cell synchronization used bar1Δ strains, and

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FACS analysis (34) was performed using a CyAn ADP analyzer (Beckman Coulter) and the FlowJo

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software package (Tree Star, Ashland OR). As both the mcm2DENQ rad9∆ and mcm2DENQ rad9∆

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sml1∆ strains quickly develop methyl methanosulfonate (MMS)-resistant variants, these strains were

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routinely retested for MMS sensitivity prior to experimentation. As no phenotypic differences could be

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observed between these two strain backgrounds, they were used somewhat interchangeably throughout

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as detailed in the figure legends.

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Immunological methods. Tubulin immunofluorescence was performed as described (35) using a rat

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anti-tubulin primary antibody (YOL 1/34, Serotec) and an Alexa Fluor 546 goat anti-rat IgG secondary

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(A11081, Invitrogen). In this assay, spindles confined to the mother cell were scored as “short” (pre-

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mitotic) while those spanning both nascent daughter cells were scored as “long” (post-mitotic). Co-

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immunoprecipitation (36) experiments between Mrc1-3XHA, Csm3-3XHA and Mcm2-7, were

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conducted as previously described (25), except that the media was supplemented with 2% raffinose

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during galactose induction. Antibody incubation of the extract was carried out at 4o C with anti-HA

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(HA.11, Covance) or one of several pan-Mcm antibodies as indicated (monoclonal AS 1.1 (30) or

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polyclonal rabbit UM174 (S.P. Bell, MIT)) for two hours followed by a one-hour incubation with

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GammaBind Sepharose protein G beads (GE Healthcare). FLAG co-IPs were performed as previously

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described (37) using FLAG-M2 agarose beads (Sigma).

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For Mrc1, Rad9, and Rad53 phosphorylation assays and Mcm quantitative western blot

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analyses, protein extracts were prepared by the trichloroacetic acid (TCA) precipitation method, and

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the corresponding blot was visualized using chemiluminescence (Femto kit, Pierce) and a Fuji LAS-

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3000 CCD system in conjunction with Image Gauge software. The following Santa Cruz antibodies

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were used: anti-Mcm2 (SC-6680), anti-Mcm5 (SC-6686), and anti-Rad53 (SC-6749). Additional

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antibodies used: FLAG M2 (Sigma, F1804); anti-G6PDH (Sigma, A9521). Mcm4 and Mcm6 were

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visualized using monoclonal antibodies AS6.1 and AS3.1 respectively (A. Schwacha and S.P. Bell,

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unpublished data, (30)).

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Mcm2-7 protein stability and limited proteolysis. Cycloheximide chase experiments were

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performed as previously described (36). Briefly, cycloheximide was added to exponentially growing

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cells to a final concentration of 50 μg/ml at time zero. Culture aliquots were withdrawn at indicated

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times and subjected to extract preparation and western blot analysis. Wild type and Mcm2DENQ

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Mcm2-7 complexes were purified from baculovirus-infected insect cells (32), and subjected to limited

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proteolysis (38). Both protein preps were extensively characterized for subunit stoichiometry by

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quantitative western blotting following Mcm4 immunoprecipitation (minimal hexamer content for wild

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type Mcm2-7 preparation = 81%; for Mcm2-7 preparation containing Mcm2DENQ = 59%). To

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perform limited proteolysis, 2 pmol of purified protein was incubated in S/0.1 buffer (33) containing

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2.5μg/ml trypsin, 10mM MgOAc, and 10mM ATP (as indicated) in a final volume of 5μl. At each

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indicated time point, kill cocktail (1.6X SDS loading dye, 6.67mM PMSF, and 6.67μg/ml TPCK in a

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total volume of 15μl in S/0.1 buffer (33) was added to stop the reaction, and the tube was incubated on

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ice. Proteins were separated on a 7% SDS-PAGE gel and visualized by silver staining.

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Sister chromatid cohesion assay. The SCC assay used a Lac operator array integrated into

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chromosome IV at position 932,137 (39). Cells were arrested at G1 with α-factor for 3 hours, washed,

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and released into fresh YPD containing 15μg/ml of nocodazole for 2 hours. The number of cells

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displaying two separate GFP dots in close proximity was scored using a Zeiss Axioskop 40 microscope

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and ≥100 cells were scored for each time point.

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Genomic Methods. Chromatin immunoprecipitation (ChIP) was performed as described (21, 34)

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using yeast engineered to facilitate Bromo deoxyuridine (BrdU) uptake (40). For experiments to

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examine Mcm localization in G1, cells were arrested with α-factor at 23oC for 3 hours, and

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subsequently processed for ChIP-Seq, using either a pan-Mcm2-7 polyclonal antibody (UM174, S.P.

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Bell, MIT) or anti-Mcm2 (Santa Cruz SC6680) as indicated. Both treatments were highly concordant

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and exhibited more than 92% overlap between peaks detected with each antibody from either wild type

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or mcm2DENQ. Cells used for BrdU ChIP-seq were similarly α-factor arrested, but released into YPD

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media containing 200 mM HU and 400 μg/ml BrdU for 100-110 minutes at 23oC (depending upon the

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replicate, see Supporting Information), and subsequently processed for ChIP-Seq using an anti-BrdU

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antibody (555627 BD Bioscience). Sequencing libraries were generated from the corresponding

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immunoprecipitated DNA using the Illumina sample prep kit, multiplexed, and sequenced on a GAII

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Illumina sequencer. The resulting data were processed, assembled, and normalized using standard

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methods with SCS2.6 software (Supporting information). Approximately five million reads per

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experiment were obtained. All genomic experiments were performed in duplicate; for presentation

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purposes these replicates were combined following quantile normalization as indicated. Details of the

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genomics procedure and analysis may be found in the Supporting information, while the primary

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genomics data from this paper is available from the Gene Expression Omnibus (GEO;

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http://www.ncbi.nlm.nih.gov/geo/) database as record number GSE38032. (Reviewer note – the above

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accession number is non-functional until publication. Contact the editor to access this data for review

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purposes).

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Results

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Preliminary Considerations and Experimental Rationale. The role of Mcm2-7 in the DRC was

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probed using the mcm2DENQ allele, a substitution of the two universally conserved acidic residues of

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the Walker B ATPase motif for their amide counterparts (DENQ). Biochemically, the mcm2DENQ

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mutation abolishes ATP hydrolysis at the Mcm6/2 active site; however, in the context of the complete

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Mcm2-7 complex, it has little or no effect on in vitro DNA unwinding (32, 33). Interestingly it is one

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of the few mcm mutations among many examined that demonstrate defects related to Mcm2/5 gate

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opening (32). We therefore focused on the effects of mcm2DENQ for DRC checkpoint activation and

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signal transduction.

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As indicated, the mcm2DENQ allele was compared to other yeast mcm alleles with more

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generic defects. One was mcm4F391, a hypomorphic allele analogous to the mouse mcm4Chaos3

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mutation previously shown to cause mammary adenocarcinomas (referred to hereafter as

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mcm4Chaos3) (41)(42); the corresponding yeast mutation also confers genomic instability (43). Two

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other viable reference MCM alleles that each ablate the Mcm4/6 ATPase site were also examined; the

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Mcm4 arginine finger mutant allele (mcm4RA) and the Mcm6 Walker B mutant allele (mcm6DENQ)

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have been previously analyzed biochemically and, in contrast to mcm2DENQ allele, both demonstrate

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near normal Mcm 2/5 gate conformation (32, 33).

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The mcm2DENQ mutant has a classic DRC phenotype. In wild type cells, hydroxyurea (HU)

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generates replication stress and activates the DRC. In contrast, exposure to the radiomimetic chemical

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MMS generates DNA damage and activates the DDC. In yeast both signal transduction cascades

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depend on Mec1 as the sensor, and on Rad53 as the terminal effector (reviewed in (44)), but utilize

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different mediator proteins as the immediate targets of Mec1 phosphorylation; the DRC is specifically

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dependent upon Mrc1 (3) while the corresponding DDC mediator is Rad9 (45).

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The mcm2DENQ mutant has phenotypes similar to mrc1 mutant alleles, but different to those conferred by rad9Δ, implying specific loss of DRC function: (1) In general, DRC mutants (e.g., mrc1Δ) are specifically sensitive to HU; while DDC mutants

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(e.g., rad9Δ) are specifically sensitive to MMS (4, 10, 46). In the absence of either chemical, the

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mcm2DENQ mutant exhibited a nearly normal colony size. However, in the presence of a moderate

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concentration of HU the mcm2DENQ mutant grew slowly and formed small colonies (Fig. 1A), but

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was relatively resistant to MMS (Fig. 1B), consistent with a defect in the DRC but not the DDC.

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Unlike the mcm2DENQ mutant, the mcm reference mutants produce robust colonies in the presence of

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HU or MMS, implying that their checkpoint response is normal (Fig. 1B, data not shown).

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(2) Induction of either a DRC or a DDC response ultimately results in Rad53 phosphorylation

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and its activation, which triggers a checkpoint response via the subsequent phosphorylation of

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numerous downstream targets (reviewed in (44)). However, there is significant cross-talk between the

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two responses (47)(48) and their partial overlap causes diagnostic mutant phenotypes. First, single

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DRC- or DDC-specific mutants (e.g., mrc1Δ or rad9Δ) still exhibit near wild type resistance to the

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orthologous challenge, i.e., MMS or HU respectively (Fig. 1A, B). In contrast, the presence of both

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mutations synergistically results in extreme sensitivity to either chemical (Fig. 1A, B). [It should be

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noted that although simultaneous loss of both the DDC and DRC is lethal (e.g., mec1Δ, rad53Δ, and

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the rad9Δ mrc1Δ double mutant (3, 49, 50)), such lethality can be suppressed by deletion of the SML1

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gene (51) which is included in our strains as indicated].

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In analogous double mutant combinations, the mcm2DENQ mutation confers the same effects

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as canonical DRC-defective alleles (Fig. 1B). First, the mcm2DENQ rad9Δ double mutant was

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synergistically sensitive to MMS exposure. Second, a combination of mcm2DENQ with mrc1AQ, a

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non-phosphorylatable DRC allele (4), resulted in the same MMS sensitivity as the corresponding

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single mutants. [We note that the mcm2DENQ mrc1Δ double mutant is inviable and could not be

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tested]. Several additional observations suggest that the MMS-sensitivity in the mcm2DENQ rad9Δ

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double mutant stems from loss of ATP hydrolysis at the Mcm6/2 active site. This phenotype does not

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arise from a general reduction in mcm2DENQ protein levels, as over-expression of this allele provided

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no substantial increase in MMS resistance (Fig 1B, bottom), Moreover, this DRC phenotype was

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specific to the mcm2DENQ allele, as none of our other reference alleles, either by themselves or in

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combination with rad9Δ, caused increased MMS sensitivity (Fig. 1B, data not shown). Therefore, we

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infer that a DRC defect does not simply arise as a secondary consequence of a general mcm defect. As

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the mcm2DENQ rad9Δ double mutant is considerably more sensitive to MMS that HU, most of the

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following experiments will examine the effects of MMS rather than HU unless specified.

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(3) The DDC and DRC both block cell cycle progression in response to MMS-induced DNA

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damage. In the DDC-defective rad9Δ mutant, cell cycle progression continues in the presence of DNA

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damage after a significant lag (52). This lag reflects a concomitant activation of the DRC (above) and

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thus is eliminated by DRC-specific mutations. Thus, simultaneous loss of both checkpoint pathways

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results in a synergistic increase in aberrant cell cycle progression in the presence of MMS.

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By this criterion the mcm2DENQ mutant exhibits the same phenotype as canonical DRC

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mutants upon MMS exposure. To assay inappropriate mitotic entry, we examined spindle elongation

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using tubulin immunofluorescence (Fig. 1C). As previously noted (4), wild type cells grown in the

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presence of MMS arrest with short spindles that do not extend into the bud, whereas 10-20% of cells

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containing either a rad9Δ or a mrc1Δ mutation attempt cell division and acquire long spindles

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spanning the bud (Fig. 1D). These two mutations function synergistically, as nearly 50% of rad9Δ

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mrc1Δ sml1Δ cells acquire long spindles under these conditions (Fig. 1D), indicating abnormal mitotic

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entry. The mcm2DENQ rad9Δ (sml1Δ) double mutants similarly acquired long spindles, nuclear

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fragmentation, and inviability (Fig 1C-E), albeit with somewhat slower kinetics than a canonical DRC-

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DDC mutant (e.g., rad9Δ mrc1Δ sml1Δ).

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Additionally, like many checkpoint mutants, FACS analysis demonstrates that the mcm2DENQ

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mutant grew more slowly than the wild type strain during unchallenged growth (Fig. 1F) with slightly

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prolonged S (~10 minutes) and G2 phases (~10-20 minutes). Despite the slow growth, the mcm2DENQ

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mutant by itself has a normal response to DNA damage; in the presence of MMS, the wild type and

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mcm2DENQ strains both proceed very slowly through S-phase with identical kinetics (Fig. 1F). Thus,

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the mcm2DENQ mutant has an essentially normal cell cycle response to DNA damage, reflecting the

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functional redundancy between the DDC and DRC pathways.

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However, similar to other DRC alleles, the mcm2DENQ allele demonstrates a synergistic loss

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of checkpoint control when ablated for the DDC mediator rad9∆. Although the rad9∆ mutant by itself

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only partially eliminated the MMS-induced block to S-phase progression, the mcm2DENQ rad9∆

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double mutant almost completely eliminated the MMS-induced block to S-phase progression and

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proceeded through the cell cycle identically in either the presence or absence of MMS (Fig. 1F).

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Taken together, the above mcm2DENQ phenotypes imply that Mcm2-7 has a specific role in the DRC but no discernible role in the DDC.

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The mcm2DENQ mutant demonstrates normal origin loading but defective late origin firing. To

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better understand the relationship between the DRC and replication, we assessed the replication ability

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of the mcm2DENQ mutant. DRC mutations (e.g., in MEC1 or MRC1) inappropriately activate late

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firing origins (21, 53) in the presence of replication stress. In contrast, specific loss of the DDC (i.e.,

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rad9Δ) results in little or no change in origin firing under these conditions (53).

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We first tested the mcm2DENQ mutant for obvious replication defects. A primary requirement

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of replication is that Mcm2-7 properly associates with replication origins during G1 phase (pre-

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replication complex formation, reviewed in (54)). Toward this end, we assessed the localization of the

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Mcm2-7 helicase at origins in G1 from wild type and mcm2DENQ strains by ChIP-Seq. We identified

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377 genomic Mcm-association sites in the mcm2DENQ mutant (Fig. 2A). Nearly all such sites were

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qualitatively similar and concordant with those previously published for the wild type strain (Fig. 2A,

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oriDB, (55)). Thus, the mcm2DENQ mutant appeared to be essentially normal for Mcm2-7 origin

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loading.

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To examine origin firing in the mcm2DENQ mutant, we modified our ChIP-seq approach to

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enrich for nascent DNA fragments containing newly-incorporated bromo deoxyuridine (BrdU)(56).

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We observed that during S-phase, the sites of BrdU incorporation in wild type, mrc1∆, and

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mcm2DENQ strains mapped to sites of pre-RC formation at previously identified origins (Fig. 2B)

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(55). However, the origin subsets activated in the presence of HU in mcm2DENQ, and mrc1∆ differed

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significantly, and similarly, from those observed in wild type (Fig. 2B).

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By comparison to prior origin utilization data (53), we found no significant quantitative

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differences in BrdU incorporation at early origins among the three strains (Fig 2C1). However in

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contrast, we observed as expected a marked increase in BrdU incorporation at late origins in the mrc1∆

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strain (Fig 2C2). The mcm2DENQ strain exhibited an intermediate phenotype with significantly more

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BrdU incorporation at this subset of late origins than in wild type (Fig. 2B). Previous studies have

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shown that different DRC mutants (e.g., mrc1AQ) activate different arrays of late origins (53).

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Comparison of BrdU incorporation between mrc1AQ specific origins and all late activating origins

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(i.e., those activated in mrc1∆) revealed a significant increase in BrdU incorporation in the

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mcm2DENQ mutant (Fig. 2C3, p-value < 0.0001). In contrast, the mcm2DENQ mutant demonstrated

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no significant enrichment of BrdU in the set of ‘all late origins minus mrc1AQ origins’ that fire in

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mrc1∆ but not mrc1AQ (Fig. 2C4, p-value 0.4). Together, these results underscore the similarities

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between the mcm2DENQ mutant and established DRC mutants.

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The mcm2DENQ mutant can sense replication stress, but is partially defective in Rad53

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phosphorylation. In contrast to the reference mcm alleles, we demonstrate that the mcm2DENQ mutant

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is defective in the DRC checkpoint response to HU-induced replication stress. In principle, such a

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defect could presumably result from a trivial failure to generate the requisite ssDNA signal, either due

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to a reduced number of replication forks or a partial defect in DNA unwinding (14, 15). However, a

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more interesting alternative is that the defect arises from disruption of some critical checkpoint

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function occurring downstream of Mec1 activation that requires a direct involvement of Mcm2-7 in the

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DRC cascade.

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To distinguish between these two possibilities, we investigated the effects of mcm2DENQ on

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phosphorylation of Mrc1 and Rad53 in response to HU or, as a control, MMS. As described above,

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either challenge activates both the DRC and the DDC, albeit to different relative extents. Members of

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the cascade are activated through phosphorylation, which can be easily assayed via mobility shifts

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observed following western blotting (4, 57, 58).

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In wild type cells under replicative stress, Mrc1 is phosphorylated by Mec1. This, in turn, is

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required for Rad53 activation and is a diagnostic indicator of initial DRC activation (3, 4). Although

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we did not examine of the kinetics of Mrc1 phosphorylation following exposure to replication stress,

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we observed identical steady-state levels of Mrc1 phosphorylation in both the wild types strain and the

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mcm2DENQ mutant upon HU treatment. Moreover, identical results were observed for the

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mcm2DENQ rad9∆ double mutant in HU, where any possible contribution of the DDC is eliminated

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(Fig. 3A). Previous reports indicate that under certain conditions Mrc1 can be phosphorylated by either

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the Mec1 or Tel1 kinases (59). We found that neither tel1∆ sml1∆ nor the mcm2DENQ tel1∆ sml1∆

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mutants block Mrc1 phosphorylation, while a mec1∆ tel1∆sml1∆ triple mutant completely eliminated

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Mrc1 phosphorylation (Fig 3A). [Note: the mcm2DENQ mutant is inviable in combination with mec1∆

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sml1∆].

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Thus, the mcm2DENQ mutant is able to initially recognize and initiate early stages of the DRC

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cascade in response to replication stress, strongly suggesting that the DRC defect in this mutant is not a

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trivial consequence of insufficient ssDNA production during replication stress.

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The mcm2DENQ mutant must therefore be defective at a later stage of the DRC. To examine

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this possibility, we next assayed Rad53 phosphorylation in response to either HU or MMS. The

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wildtype strain, mcm reference alleles, and single mutants defective in either the DRC or DDC all still

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exhibited robust Rad53 phosphorylation in response to either HU or MMS (40-60% of Rad53 in one of

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several higher molecular weight forms), while double mutants that eliminate both pathways (mrc1∆

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rad9∆ sml1∆) completely eliminated phosphorylation (Fig. 3BC). Furthermore, none of the reference

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mcm alleles showed any reduction in Rad53 phosphorylation when combined with a rad9∆ mutation,

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in the presence of either MMS or HU (Fig. 3C), indicating proficient DRC activation and

343

implementation (above). In contrast, the mcm2DENQ mutation conferred the same phenotypes as

344

canonical DRC mutations in this test. We observed robust phosphorylation in the mcm2DENQ single

345

mutant and a 2-3 fold decrease in Rad53 phosphorylation in the mcm2DENQ rad9∆ double mutant.

346

Moreover, a double mutant containing both mcm2DENQ and a canonical DRC mutation (mrc1AQ)

347

exhibited only a modest reduction in Rad53 phosphorylation similar to the respective single mutants

348

(Fig. 3B), consistent with Mrc1 and Mcm2-7 functioning in a common pathway. Finally, in the

349

presence of MMS, both the mcm2DENQ mutant and a mcm2DENQ/mrc1AQ double mutant

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350

demonstrated robust Rad9 phosphorylation (Fig. 3D), confirming normal integrity of the DDC cascade

351

in this strain.

352

In summary, these results indicate that the DRC phenotype of the mcm2DENQ mutant is not

353

caused by a generic replication defect but rather by a specific defect in the checkpoint cascade,

354

presumably after Mec1 activation and Mrc1 phosphorylation, but prior to Rad53 activation. Thus, in

355

addition to its previously-described roles, (60, 61) Mcm2-7 has an independent specific role in DRC-

356

mediated signal transduction.

357 358

The mcm2DENQ mutant maintains association among Mcm2-7, the DRC mediator proteins and

359

CDC45. Although our data indicates that faulty DNA replication is not responsible for the

360

mcm2DENQ DRC phenotype (above), other indirect causes for this phenotype were also considered.

361

One such possibility is that within the mcm2DENQ mutant, Mcm2-7 fails to physically interact

362

with the DRC mediators Mrc1, Tof1 or Csm3 (19, 20, 62). To test this possibility, we performed

363

reciprocal co-immunoprecipitation experiments (Fig. 4A). In the mcm2DENQ mutant, we found that

364

the physical association between the Mcm complex and Mrc1 was robust and maintained at nearly

365

wild type levels in both asynchronous culture (Fig. 4A) as well as during replication stress (Fig. 4B).

366

As Mrc1 association with Mcm2-7 has been reported to require Tof1 and Csm3 (19), the robust

367

physical association between Mrc1 and Mcm2-7 in the mcm2DENQ mutant strongly implies that these

368

other two checkpoint mediators properly interact with Mcm2DENQ-containing Mcm2-7 complexes.

369

Our results confirm that Tof1 loss greatly reduces Mrc1 association with Mcm2-7, implying that

370

physical association between Mcm2-7 and Tof1 is robust in the mcm2DENQ mutant (Fig. 4D). In

371

contrast with the tof1Δ mutant, loss of Csm3 appears to cause little change in Csm3-Mcm2-7

372

association (Fig. 4D). However, co-IP experiments between Mcm2-7 and Csm3 detect an equivalent

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373

interaction in both the wild type and mcm2DENQ strains (Fig. 4C) suggesting that Csm3 is not needed

374

for the association between Mrc1 and Mcm2-7 (Fig. 4D). Thus, the physical association between

375

Mcm2-7 and both Mrc1 and Csm3 is uncompromised by the mcm2DENQ mutation.

376

We next examined whether the mcm2DENQ mutation destabilizes the CMG complex during

377

replication stress (Fig. 4E). In the presence of HU, during which the CMG complex would be expected

378

to normally remain intact, Cdc45 remains associated with Mcm2-7 in both the wild type and

379

mcm2DENQ strains. In contrast, under conditions were the CMG complex is inactive (e.g., during G1

380

or G2), Cdc45 loses Mcm2-7 association in both the wild type or mcm2DENQ strains. Thus, the

381

mcm2DENQ mutation appears to cause little or no instability of the CMG complex in the presence of

382

replication stress.

383

In summary, these results largely rule out the possibility that the mcm2DENQ DRC phenotype

384

is due to a gross inability to recruit either the DRC mediators or Cdc45 to forks or to maintain their

385

physical association during replication stress.

386 387

The mcm2DENQ DRC defect is not attributable per se to a loss of sister chromatid cohesion

388

(SCC). SCC involves the stable physical association between sister chromatids during G2 (reviewed in

389

(63)). Most DRC mutants have associated SCC defects (64, 65), raising the possibility that that the

390

DRC defect in the mcm2DENQ mutant is a secondary consequence of faulty cohesion. We examined

391

this using a cytological assay that visualizes a specific test chromosome region marked by a lac

392

operator array bound to a GFP-LacI fusion protein (39).

393

We first examined SCC during G2 arrest in the presence of nocodazole (Fig. 4F). In contrast to

394

wild type cells (~2% cells with an SCC defect), ~ 35% of cells in a cohesin establishment mutant

395

(eco1-1, (66)) contain two GFP foci foci, indicative of a severe SCC defect. The mcm2DENQ mutant

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396

demonstrates an intermediate but significant defect in SCC relative to the wild type strain (p=0.004),

397

with a seven-fold increase in defective SCC similar in magnitude to that previously observed in a

398

mrc1∆ mutant (mcm2DENQ vs. mrc1Δ, p=0.09) (65). In common with its DNA damage sensitivity

399

phenotype (Fig. 1B), overexpression of the mcm2DENQ allele is unable to significantly suppress this

400

SCC phenotype, indicating that this defect is not due to a quantitative loss of Mcm2 protein.

401

Furthermore, we find that both the mcm4 Chaos3 (p=0.07) and mcm6DENQ (p=0.001) mutants lacking

402

obvious checkpoint defects also demonstrate an SCC defect relative to the wildtype strain, although the

403

difference between mcm4 Chaos3 and wild type lacks statistical significance (Fig. 4F). In contrast, the

404

mcm4RA mutant has essentially normal SCC relative to the wildtype strain (p=0.2).

405

In summary, these data fail to show an obligatory connection between defects in the DRC and

406

SCC. In contrast to the DRC defect, the SCC defect appears to be a general phenotype common to

407

diverse MCM alleles. Formally this finding implies that an SCC defect is insufficient to activate the

408

DRC and thus is not per se the unique cause of the mcm2DENQ DRC defect. It remains to be

409

determined why checkpoint-defective mutants exhibit an SCC defect and, as a separate effect, why

410

SCC is also abrogated by canonical mcm alleles that do not activate the checkpoint.

411 412

The mcm2DENQ allele demonstrates little or no off-target defects. Amino acid substitutions often

413

cause collateral defects in protein expression, folding or stability (e.g., (67)). We examined these

414

issues to determine if they were sufficient to explain the mcm2DENQ phenotypes. Potential collateral

415

defects of this allele on Mcm2 protein expression or stability were examined and ruled out (Fig. 5AB).

416

Moreover, co-immunoprecipitation experiments indicated that the allele has essential no effect on

417

assembly or stability of the Mcm2-7 complex in vivo (Fig. 5C), while limited proteolysis experiments

418

using recombinant Mcm2-7 complexes demonstrated that the mcm2DENQ mutation imparts little

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419

apparent change to the in vitro conformational stability of the Mcm2-7 complex (Fig. 5D). In contrast,

420

parallel experiments that measure the expression and stability of the mcm6DENQ and mcm4RA

421

proteins demonstrate a slight reduction of expression (both alleles) and stability (mcm6DENQ) (Fig. 6).

422

Therefore, we surmise that the DRC defects observed with the mcm2DENQ mutant are unlikely to be

423

caused by a quantitative lack of Mcm2-7.

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424

Discussion

425

We show that Mcm2-7 is an integral component of the DRC signal transduction cascade, acting

426

essentially at the same step as the Mec1/ATR-mediated phosphorylation of Mrc1. This activity

427

specifically requires the Mcm6/2 ATPase active site that has been previously implicated as having a

428

regulatory rather than DNA unwinding role in Mcm2-7 (32, 68, 69). Correspondingly, reference mcm

429

alleles that do not affect the Mcm6/2 active site have apparently normal checkpoint signaling (Fig. 1B,

430

3C, 6). These results confirm and extend previous findings that implicated Mcm2-7 in the DRC

431

response to MMS-induced damage but did not detect a role in HU-induced replication stress (25).

432

Our previous studies showed that the Mcm6/2 ATPase site is involved in regulating the

433

opening and closing of the Mcm DNA gate, with the mcm2DENQ mutation biasing the gate into a

434

closed conformation (32). By implication, the role of Mcm2-7 in activating the DRC involves

435

modulation of the Mcm gate and, more specifically, an open form of the gate, either statically or via

436

cyclic opening and closing. Additional considerations discussed below suggest that direct involvement

437

of Mcm2-7 in the DRC reflects a broader role for this molecular complex as an important functional

438

connection between the DRC and DNA replication.

439

As Mcm2-7 is highly conserved among all eukaryotes (reviewed in (27)), its direct

440

participation in the budding yeast DRC implies a role in the prevention of genomic instabilities linked

441

to DRC defects in cancer and other diseases (70).

442 443

Involvement of the Mcm6/2 ATPase active site in the DRC response. The Mcm6/2 active site

444

biochemically functions to regulate rather than directly participate in DNA unwinding. Early work

445

showed that a specific Mcm sub-complex (Mcm467) was competent to unwind DNA (71), while

446

addition of the remaining Mcm subunits (i.e., Mcm2, 3, and 5) inhibited this activity (69, 72). In part,

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447

this inhibition depends upon conserved ATPase motifs in Mcm2 (i.e., the function of the Mcm6/2

448

active site) rather than altered oligomerization caused by Mcm2 binding to the Mcm467 subcomplex

449

(69); mutations that reduce but do not eliminate ATP hydrolysis at this site are viable but demonstrate

450

sensitivity to DNA damaging agents consistent with a potential checkpoint defect (68).

451

Moreover, the Mcm6/2 ATPase site is connected functionally and physically to the DRC.

452

Mrc1 binds the C-terminus of Mcm6 (25). MCM6 mutants that ablate this interaction are sensitive to

453

DNA damaging agents and reduce Rad53 phosphorylation in combination with the rad9Δ allele. This

454

phenotype is due specifically to loss of Mrc1 association, as the phenotype of this Mcm6 mutant can be

455

suppressed by a Mcm6-Mrc1 fusion protein (25).

456

DRC mutants are traditionally sensitive to HU (4). Interestingly, both studies above found that

457

perturbations to the Mcm6/2 site (reduction in ATP hydrolysis or loss of Mrc1 association) cause

458

greater sensitivity to MMS than HU (25, 68). To a somewhat lesser extent, the mcm2DENQ mutant

459

has similar properties (Fig. 1B). Although the basis of this effect is unknown, it is useful to consider

460

that MMS generates DNA damage ahead of the replication fork, suggesting perhaps that the Mcm6/2

461

active site is dedicated to dealing with template problems associated with DNA unwinding.

462 463

Mcm2-7 as a central coordinator of the DRC. A checkpoint response has three basic requirements:

464

sensing the presence of a lesion; transduction of that information into activation of the signaling

465

cascade; and targeting of the effector kinase to elicit the appropriate checkpoint responses. We propose

466

that Mcm2-7 is directly involved as a central coordinator of the DRC.

467

The need for such an integrative role is emphasized by the fact that in budding yeast Mec1 is

468

the sensor kinase of both the DRC and the DDC, and, similarly, Chk1/Rad53 is the ultimate

469

downstream effector kinase of both pathways, but nonetheless mediates appropriate, distinct responses

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470

in the two cases. How are appropriate specificities conferred in both the input and output stages?

471

While the two responses are distinguished by alternative mediator proteins, some uncertainty remains

472

about the source of information that actually directs the appropriate response. Mcm2-7 is directly

473

involved in the DNA replication process and, as such, is positioned to mediate DRC function.

474

Our data in combination with previous results supports an integrative role of Mcm2-7 in the

475

DRC. First, Mcm2-7 has a clear mediator function. We show that during an in vivo block to DNA

476

replication an additional component, Mcm2-7, is required for efficient effector kinase activation. This

477

block is prior to Rad53 activation but after or concurrent with Mrc1 phosphorylation (Fig 3).

478

Moreover, the mcm2DENQ allele and a phosphorylation-defective mrc1 allele (mrc1AQ) confer

479

essentially the same defect (Fig 3B), shown by epistasis analysis to occur at the same step in the signal

480

transduction process. Thus by implication, the missing mediator function is Mcm2-7.

481

Second, Mcm2-7 likely ensures that Rad53 is activated as part of the DRC to yield a sensible

482

replication-dependent response. Prior results suggest that mediators in addition to Mrc1 (and possibly

483

Csm3 and Tof1) are required in vivo to facilitate appropriate Rad53 activation. Under simplified

484

conditions using either a purified biochemical system (73) or an engineered in vivo system (74),

485

evidence indicates that the only limitation to Rad53 phosphorylation in the DRC is the physical

486

association between Mec1 and Rad53. This physical connection is greatly stimulated by

487

phosphorylated Mrc1which binds to both kinases. However, in both of these experimental systems,

488

Rad53 activation occurs efficiently even in the absence of DNA replication or damage. As normal

489

DNA replication by its very nature generates RPA/coated ssDNA known to induce the DRC,

490

additional constraints must be present in vivo to prevent inappropriate DRC activation. Our data

491

confirms this supposition, as we show that, in contrast to the simplified systems, Mcm2-7 is

492

additionally required for full Rad53 activation. As Mcm2-7 is an essential component of active

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493

replication forks, this ensures that DRC activation is replication dependent and may additionally help

494

prevent fortuitous DRC or DDC activation.

495 496

Models for Mcm2-7 involvement in the DRC signal transduction cascade. Our prior biochemical

497

evidence indicates that the mcm2DENQ mutation interferes with the ability to open the MCM gate,

498

implying that the mutant protein predominantly exists in a gate-closed conformation (32). We propose

499

that a conformational change involved in gate opening/closing directly regulates the physical

500

interactions between Rad53 and Mrc1. One specific scenario for Mcm2-7 involvement in DRC activation could be as follows: Mcm2-

501 502

7 primarily functions as a mediator in the DRC (above). In the absence of replication stress, physical

503

interaction with Mcm2-7 occludes the Rad53-interacting surface of Mrc1 (Fig 7). Upon DRC

504

activation, a conformational change in Mcm2-7, fueled by ATP hydrolysis at the Mcm6/2 active site

505

(below), relieves Mrc1 inhibition and promotes its Rad53 activation (Fig 7). As the mcm2DENQ

506

mutant is fundamentally unable to hydrolyze ATP at the Mcm6/2 active site, a reasonable prediction

507

would be that the protein fusion between Mcm6 and Mrc1, previously shown as being competent for

508

Rad53 phosphorylation in the presence of a mrc1Δ (25), would be unable to suppress the mcm2DENQ

509

checkpoint defects. However, testing this prediction complicated by our finding that the Mcm6-Mrc1

510

fusion construct unexpectedly causes lethality in a mcm2DENQ mutant (E. Tsai, pers. obs.). Our

511

hypothesis, which couples a conformational change in a motor protein to a regulatory output, is similar

512

to a recently proposed model that connects defects in Topoisomerase II to trigging of a G2 checkpoint

513

(75).

514 515

Under this scenario, we speculate that upstream activation of the DRC leads to appropriate Mcm ATP hydrolysis and a conformational change in the Mcm ring structure. This outcome then

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516

facilitates both subsequent Rad53 phosphorylation as well as helicase inactivation (Fig. 7). This

517

scenario implies that the DRC mediator proteins (particularly Mrc1) should function to regulate Mcm

518

ATP hydrolysis. While such an analysis of Mrc1 has yet to be undertaken, the human proteins that

519

correspond to the budding yeast Tof1 and Csm3 (i.e., Timeless and Tipin), have been shown to both

520

block ATP hydrolysis and DNA unwinding of the human CMG complex, while in contrast stimulate

521

the activity of the leading strand polymerases in a purified biochemical system (76).

522

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523

Acknowledgments

524

We would like to thank S.P. Bell, A. Bielinsky, J. Diffley, S. Elledge, D. Koshland, and N. Saini for

525

providing strains, plasmids or advice; R. Elbakri and C. Poth for technical assistance; K. Arndt, W.

526

Saunders, B. Tomson and former and current members of the Schwacha and MacAlpine labs for

527

helpful manuscript comments; and R. Cha and N. Kleckner for advice and personal support.

528 529

Financial Disclosure

530

This work was funded by a NIH grant to AS (RO1GM083985) and DM (R01GM104097) and

531

American Cancer Society grants to AS (RSG-05-113-01-CCG) and DM (120222-RSG-11-048-01-

532

DMC).

533 534

Competing Interest-

535

The authors declare no competing interests.

536

Abbreviations-

537

α-fac–alpha factor; BrdU–Bromo deoxuyuridine; DDC–DNA Damage Checkpoint; DRC–DNA

538

Replication checkpoint; G6PDH–Glucose 6-phosphate dehydrogenase; HU–Hydroxyurea; MMS–

539

Methyl Methanesulfonate; vec–Empty vector; WT–Wildtype

540

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Figure Legends

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Figure 1. The mcm2DENQ mutant has DRC defects. (A) Wild type (UPY464), mcm2DENQ

736

(UPY499), rad9∆ (UPY630), mrc1∆ (UPY713), and rad9∆ mrc1∆ sml1∆ (UPY715) strains were

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plated on YPD lacking (left) or containing HU (middle). (B) Strains from (A), as well as mcm2DENQ

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rad9∆ (UPY634), mcm2DENQ rad9∆ sml1∆ (UPY732), mcm2DENQ mrc1AQ (UPY758), mrc1AQ

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(UPY773), rad9∆ mrc1AQ sml1∆ (UPY745), mcm4Chaos3 (UPY638), mcm4 Chaos3 rad9∆

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(UPY788), mcm2DENQ + PGAL1-mcm2DENQ (UPY655) and mcm2DENQ rad9∆ + PGAL1mcm2DENQ

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(UPY1231) were spotted onto the indicated media as 10-fold serial dilutions to assay viability. (C-D)

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The indicated strains from (B) were arrested in G1, released into YPD + 0.01% (v/v) MMS, and

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tubulin immunofluorescence was assayed. (C) The mcm2DENQ rad9∆ cells undergo inappropriate

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nuclear segregation and spindle elongation after 4.5 hours exposure to MMS. (D) Timecourse analyses

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of spindle elongation following G1 release, data represents mean and SD of n=3 experiments. (E)

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Viability following MMS exposure. Strains and growth conditions are identical to those in (D), and the

747

cultures plated at the indicated intervals on YPD without drug to measure viability. Results were

748

normalized to the starting culture viability; mean and SD of 3 independent analyses are shown. (F)

749

FACS analysis of the indicated isogenic strains from (A). Briefly, strains were arrested as in (D) and

750

released into fresh YPD ± 0.033% MMS. Aliquots taken at the indicated times and processed for

751

FACS. After 45 (wild type) or 55 (mcm2DENQ) minutes α-factor was re-added to restrict analysis to a

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single cell cycle. Asterisks denote timepoints that represent either the apparent start (black) or end

753

(red) of S phase.

754 755

Figure 2. The mcm2DENQ mutant demonstrates inappropriate late origin firing. Growth conditions

756

are described in Materials and Methods. (A) Mcm ChIP-seq analysis of wild type (UPY493) and

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32

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mcm2DENQ (UPY524) strains during G1 arrest using the pan-Mcm antibody. A representative region

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of Chr XIV is shown, and RPKM are plotted. (B) BrdU ChIP-seq data are shown for HU (200 mM)

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arrested wild type (UPY493), mcm2DENQ (UPY524), and mrc1∆ (UPY722) and the data is plotted

760

similar to (A). (C) Box and whisker plots describing the median and quartiles of BrdU enrichment for

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wild type (UPY493), mcm2DENQ (UPY524), and mrc1∆ (UPY722) at different subsets of origins

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(‘mrc1AQ specific origins’- those that only fire in the mrc1AQ mutant), and (‘all late origins minus

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mrc1AQ origins’- those that fire in mrc1∆ but not in mrc1AQ). Significance was determined by a one-

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tailed Wilcoxon rank sum test to examine if the mutants have differences that are greater than wild

765

type. The following p-values apply, Panel 1: WT and mcm2DENQ, W = 18903, p = 0.531; WT and

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mrc1∆, W = 21619, p = 0.994. Panel 2: WT and mcm2DENQ, W= 26665, p