1. Volume 32, Issue 1, Pages 106-117 (October 2008)
Mrc1 and DNA Polymerase ɛ Function Together in Linking DNA Replication and the S Phase Checkpoint 
Huiqiang Lou, Makiko Komata, Yuki Katou, Zhiyun Guan, Clara C. Reis, Martin Budd, Katsuhiko Shirahige, Judith L. Campbell 
Molecular Cell 
Volume 32, Issue 1, Pages (October 2008)
DOI: /j.molcel
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2. Figure 1 Mrc1 Interacts with Pol2
(A) Schematic map of the full-length POL2 gene and the zinc finger region. The full-length gene, 2222 residues, is shown at the top. Pol2N, five conserved nuclease motifs, residues 288 and 501; seven partially conserved DNA polymerase motifs, residues 544 and 954, three conserved 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, 1180 and 2103; conserved zinc finger motif, 2103–2222 (end). MutA through MutI are deletions within the zinc finger domain used for mapping the interaction subdomain in Figure S1 (Dua et al., 1998).
(B) Two-hybrid assay for Mrc1/Pol2 interaction. Analyses were performed on at least three independent transformants as described previously (Edwards et al., 2003).
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3. Figure 2 Reciprocal Coimmunoprecipitation of Pol2 and Mrc1
Mrc1-13myc/Pol2-3HA (strain AC2301, Table S1) is the doubly tagged strain used to test coimmunoprecipitation. Whole-cell extracts (WCE) were prepared from asynchronous cells as described in the Experimental Procedures. Immunoprecipitation (IP) was carried out with the antibody indicated, and proteins in the WCE, the supernatant (SN), and the immunoprecipitation were determined by western blotting. WB, western blot. WCE and SN represent the same number of cell equivalents of protein. In control experiments, 10× excess of SN was also loaded to verify estimated precipitation efficiency.
(A) Coimmunoprecipitation. Lanes 1–6, Mrc1-13myc/Pol2 indicates strain Y1134 carrying myc-tagged Mrc1 and untagged Pol2 and serves as a control that anti-HA does not immunoprecipitate Mrc1 when Pol2 is untagged. Mrc1/Pol2-3HA, strain A1591, serves as a control 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 was precipitated with anti-myc, and the amount of Pol2 and Mrc1 in the respective immunoprecipitation was determined as described in the Experimental Procedures.
(B) Salt and DNase I sensitivity of coimmunoprecipitation. Lanes 1–8, wash buffers contained the 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 top of the figure were staged in the cell cycle as described in the Experimental Procedures. Asyn, asynchronous cells; G1, cells arrested with α factor; S, cells released from α factor arrest into S phase. Immunoprecipitations were carried out with the indicated antibodies. Western blots of the precipitates are shown.
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4. Figure 3 Two Independent Interactions between Pol2 and Mrc1
(A) Competition binding assays show that Pol2N and Pol2C bind Mrc1. An anti-myc immunoprecipitation was carried out on strain AC 2301, MRC1-myc/ POL2-HA, carrying plasmid pBTM116 or pBTM116 overexpressing either Pol2C or Pol2N as potential competitors for POL2-HA binding to MRC1-myc. Lanes 1–3, no plasmid; lanes 4–6, pBTM116 vector; lanes 7–9, pBTM116 expressing Pol2C; lanes 10–12, pBTM116 expressing Pol2N. Blots of IPs in were probed with anti-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 gene and the deletion mutants is shown. P, likely Mec1/Ddc2 phosphorylation sites. The indicated N-terminal mrc1 deletion mutants were tagged with FLAG epitope, and the C-terminal mrc1 deletion mutants were tagged with HA epitopes (see the Experimental Procedures and Table S1). The POL2 gene was tagged with 13myc in strains AC (C-terminal mrc1 deletions) or with 3HA in strains AC2205–2209 (N-terminal mrc1 deletions) by homologous recombination as indicated. Immunoprecipitation was carried out with anti-FLAG antibody for the N-terminal Mrc1 deletions and anti-HA antibody for the C-terminal Mrc1 deletions, and immunoprecipitates were probed with anti-FLAG and anti-HA antibody as indicated. Anti-Pol2-myc or anti-Pol2-HA western blots of immunoprecipitates reveal efficiency of Pol2 coimmunoprecipitating with the respective mrc1 mutant proteins.
(C) S phase progression of various mrc1Δ mutants is delayed. Cells were synchronized in G1 phase with α factor and then released into the cell cycle. Samples were taken for flow cytometry analysis at the indicated times.
(D) Mrc1N binds Pol2N, and Mrc1C binds Pol2C. Binding studies were carried out as in Figure 2. Strains contained the respective, chromosomally tagged mrc1 mutant, a wild-type, untagged POL2 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 on the left, and coimmunoprecipitating species detected by western blotting are indicated on the right of each panel. Rows 1 and 2, mrc1( FLAG) carrying pACT2-HA-Pol2N; rows 3 and 4, mrc1( FLAG) carrying pACT2-HA-Pol2N; row 5, mrc1-( FLAG) carrying pACT2-HA-Pol2C; row 6, mrc1( FLAG) carrying pACT2-HA-Pol2C; row 7, mrc1( FLAG) carrying pACT2-HA-Pol2C.
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5. Figure 4 Genetic Interactions between POL2 and MRC1
(A) Phenotypes of various pol2 mrc1 double mutants. These results were obtained using two complementary methods for creating and analyzing double mutants, as described in detail in the Experimental Procedures. Strain genotypes are shown in Table S1.
(B) Suppression of pol2–11 by MRC1 overexpression. 10-fold serial dilutions of exponentially growing cultures of pol2–11 cells transformed with plasmids expressing MRC1 (pACT2-MRC1, pBTM116-MRC1) or control pol2–11 carrying empty pACT2 or without plasmid were plated 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 of strain pol2–16 transformed with plasmids indicated on the left (Table S2) were grown at different temperatures or in the presence of HU as indicated.
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6. Figure 5
Regulation of Pol2/Mrc1 Interaction by Phosphorylation of Mrc1
(A) Pol2 interacts with Mrc1AQ. Anti-myc immunoprecipitation of strain mrc1AQ-myc/POL2-HA, AC2302. IPs were probed with anti-HA antibody to detect Pol2 as in Figure 2C. The experiment was carried out simultaneously with that shown in Figure 2C.
(B) Pol2 interacts with both nonphosphorylated and phosphorylated Mrc1. Mrc1-13myc and Pol2-3HA 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) was synchronized with α factor and released into fresh medium containing 75 mM HU for 60 min followed by immunoprecipitation with anti-HA antibody (see text for conditions compared). Mrc1-P refers to phosphorylated Mrc1.
(C) Mrc1-P does not interact with Pol2N but does interact with the Pol2-C. Pol2N and Pol2C were each tagged with HA in pACT2 and expressed in an Mrc1-13myc strain (Y1134) in the absence or presence of the indicated amounts of HU. HA-Pol2N or HA-Pol2C was immunoprecipitated and Mrc1 or Mrc1-P was detected in the immunoprecipitation with α-myc (rows 1–7). WB HA indicates the control for Pol2N and Pol2C immunoprecipitation efficiencies, rows 3, 5, and 7. Rows 8 and 9 are Mrc1-myc IPs. Row 9 is control for Mrc1 IP. The various rows and lanes are described in the text.
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7. 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, or tof1Δ strains were synchronized with α factor and released from pheromone arrest in the presence of 200 mM HU. After 60 min, ChIP analysis was carried out as described previously. The blue histograms represent Pol2 enrichment. The vertical axis is enrichment on a log2 scale. The horizontal axis is length in kilobases.
(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 for coimmunoprecipitation (strains listed in Table S1). Whole-cell extracts (WCE) were prepared from asynchronous cells (Asyn), cells arrested with α factor (G1), or cells released from α factor into S phase in the presence of HU treatment (HU). Immunoprecipitation was carried out as described in the legend to Figure 3 and in the Experimental Procedures. Lanes 1–3 show a control strain with untagged Mcm2. Lanes 1–3, top row, show that Mcm2 was not immunoprecipitated by the myc antibody. Lanes 1–3, bottom row, show that the Mrc1-FLAG did not immunoprecipitate with the myc antibody in the absence of myc-tagged Mcm2. Control showing that Pol2-HA does not coimmunoprecipitate with myc antibody is shown in Figure 2A, lane 6.
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8. Figure 7
Models of Functional Mrc1/Pol2 Interaction on the Leading Strand
(A) Interaction during normal DNA replication. The shape of pol ɛ depicted is derived from reference (Asturias et al., 2006). The interactions between Mrc1 and Pol2 described in this study are incorporated into a rudimentary model for polymerase/helicase coupling during normal DNA replication (see text).
(B) Mrc1/Pol2 interaction on the leading strand during replication stress. Pol ɛ/Mrc1 senses the damage and phosphorylation of Mrc1 releases the N terminus, but there is continued interaction between Mrc1-P with the C terminus of Pol2.
Molecular Cell  , DOI: ( /j.molcel )
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