HR proteins have a role in recovery from HU distinct from that in the checkpoint. Asynchronous cultures of wild-type (FY72), Δcds1 (FY865), Δrad3 (FY1106), Δrad22 (FY1209), and Δrhp51 (FY1884) cells grown at 25°C were treated with 15 mM HU for 4 h, then released into fresh medium without HU, and grown for another 4 h at 25°C. Cells were analyzed for (A) DNA content by flow cytometry and (B) nuclear morphology by staining with Sytox green. Arrowheads indicate examples of chromosome missegregation events (detailed in the text); averages and standard deviations were calculated from three independent experiments. Scale bar, 10 μM.

MCM proteins interact with Rad51 in HeLa cells. (A) Whole-cell lysates prepared from asynchronous cultures of HeLa cells (asynch), cells blocked in 2 mM thymidine for 18 h and then released into S phase for 3 h (S phase), and cells blocked in early S phase with 2.5 mM HU for 18 h (HU) were immunoprecipitated with anti-Rad51 antibodies or with an irrelevant, control antibody (anti-myc). The anti-myc immunoprecipitation (IP) was performed on cells blocked in HU. Rad51 and associated proteins were analyzed by 10% SDS-PAGE, followed by immunoblotting to detect Rad51 and Mcm7. (B) Coimmunoprecipitation of MCM7 with Rad51 from HeLa cells is not dependent on DNA. Anti-Rad51 antibodies immunoprecipitate Rad51 and MCM7 from both untreated HeLa cell lysates and lysates that were treated with ethidium bromide (EtBr). AS, asynchronous cells; S, cells released from thymidine block into S phase; HU, cells arrested in hydroxyurea. C, control IP (anti-myc).

MCM proteins are required for recovery from HU. (A) Schematic of the experimental protocol. Wild-type (FY72), mcm4ts (FY364), and mcm4ts-degron (FY2887) cells were grown asynchronously (white arrows) and then arrested in HU for 4 h (black arrows). Cells were shifted to the restrictive temperature of 36°C and then released from HU. Samples were taken at time points t1, t2, t3, and t4. (B) Cellular DNA content was analyzed by flow cytometry. All strains contained approximately 1C DNA content in HU. The wild-type and mcm4ts cells, but not the mcm4ts-degron cells, completed bulk synthesis upon release from HU. (C) Inactivation of Mcm4 resulted in DNA breaks in HU. Wild-type and mcm4ts-degron mutants were arrested in HU at 25°C (t2) and shifted to 36°C while maintained in HU (t3). DNA DSBs were detected by phospho-H2A immunostaining as described in the legend to Fig. 1. (D) Cellular morphology was examined by staining with Sytox green to detect the DNA. Scale bars, 10 μm.

mcm-ts mutants generate DNA damage during replication. (A) mcm4ts cells generate DNA damage. Spread nuclei prepared from wild-type (FY72) and mcm4ts (FY364) cells shifted to the restrictive temperature (36°C) for 4 h were immunostained for phospho-H2A (green) to mark DNA breaks and counterstained with DAPI (blue) to detect the DNA. α-pH2A, anti-phospho-H2A antibody. Scale bar, 10 mM. Two examples of wild-type nuclei are shown; while most nuclei did not contain phospho-H2A foci, a small number of S-phase nuclei displayed a few phospho-H2A foci. (B and C) DNA damage in mcm4ts cells requires S-phase progression. Wild-type (FY72), mcm4ts (FY364), and mcm4ts-degron (FY2887) cells grown at 25°C were blocked in G₁ by nitrogen starvation and then released into S phase at 36°C. At 1-h intervals, (B) DNA content was measured by flow cytometry, and (C) DNA damage was examined by immunostaining spread nuclei for phospho-H2A. (D) DNA damage in mcm4ts cells corresponds to sites of new synthesis. Wild-type (FY2317) and mcm4ts (FY2514) cells were shifted to the restrictive temperature for 3.5 h and then pulse-labeled with BrdU for 30 min. Stretched chromatin fibers were then prepared from these cells and visualized with DAPI (left). Chromatin fibers were immunostained with antibodies to BrdU and phospho-H2A to detect sites of replicating DNA and DNA breaks, respectively (right). Scale bar, 10 μm (approximately 18 kb).

siRNA against MCM4 or MCM7 in HeLa cells generates DNA damage and cell inviability. HeLa cells were transfected with buffer only (mock) or with siRNA against MCM4 or MCM7, and the cells were analyzed at 0 and 48 h after transfection. (A) Proteins were analyzed by 10% SDS-PAGE, followed by immunoblotting (IB) with antibodies to detect MCM4 or MCM7, and subsequently immunoblotted with anti-PCNA antibody as a loading control. (B) DNA content was assessed by flow cytometry. HeLa cells mock treated or treated with MCM4 siRNA or MCM7 siRNA for 48 h were stained with (C) DAPI to visualize the DNA, and (D) phospho-H2AX and DAPI to detect DNA damage. (E) The DNA damage correlates with loss of MCM localization and corresponds to sites of new DNA synthesis. Stretched chromatin fibers prepared from HeLa cells 48 h after siRNA treatment were immunostained for MCM7 and phospho-H2AX and counterstained with DAPI (left); chromatin fibers prepared from cells pulse-labeled with BrdU for 1 h were immunostained for BrdU and Rad51 and counterstained with DAPI. Scale bar, 10 μm (approximately 20 kb). (F) MCM siRNA results in cell death by apoptosis. Cells mock treated or treated with siRNA against MCM4 or MCM7 were immunostained for cleaved caspase 3 and counterstained with DAPI. Scale bars, 10 μm.

Cds1-dependent phosphorylation of Mcm4. (A) Mcm4 coimmunoprecipitates Cds1. Protein lysates prepared from wild-type (FY72), mcm4-HA (FY1167), mcm4-HA Δrhp51 (FY2356), cds1-myc (JBY267), and mcm4-HA cds1-myc (JBY235) strains incubated with 15 mM HU for 4 h were immunoprecipitated (IP) with anti-HA antibodies or control (C) antibody (antitubulin); immunoprecipitates were then immunoblotted (IB) to detect Cds1-myc and Mcm4-HA. (B). Schematic of the experimental protocol. Cells were grown asynchronously (white arrow) and arrested in HU for 4 h (black arrow). Cells were then released into medium with or without HU, either at 25°C or 36°C. Samples were taken at time points t1, t2, t3, t4, t5, and t6. (C) Cellular DNA content was analyzed in wild-type (FY7), mcm4ts (FY1413), Δcds1 (FY2423), Δcds1 mcm4ts (FY1617), and mcm4ts-degron (FY3396) cells by flow cytometry. (D) Relative viability of the strains in panel C was determined as described in Materials and Methods. (E) Mcm4 gel mobility shift in HU. Trichloroacetic acid-precipitated lysates from the strains indicated in panel C were immunoblotted (IB) for Mcm4 protein with anti-Mcm4 antibody (α-Mcm4). The shifted (phosphorylated [P]) form of Mcm4 is indicated by the white arrowhead. The Mcm4-degron protein exhibits reduced mobility relative to wild-type Mcm4 because of the large N-terminal degron tag. Strain FY2423 contains Mcm4HA, but identical results were observed using FY866, which has untagged Mcm4 (data not shown).

Recombination proteins in the HU response (A) Mcm4 coprecipitates Rhp51 in HU. Protein lysates prepared from wild-type (FY72), mcm4-HA Δrhp51 (FY2356), and mcm4-HA (FY1167) cells untreated or treated with 15 mM HU for 4 h (+HU) were immunoprecipitated (IP) with anti-HA antibodies, an irrelevant control antibody (lane C), or protein A beads only (lane B). Immunoprecipitates and whole-cell lysates were analyzed by 10% SDS-PAGE, followed by immunoblotting (IB) with antibodies to detect Mcm4-HA (anti-HA) and Rhp51 (anti-Rhp51). (B) HR mutants display a modest loss of viability in HU compared to checkpoint mutants. Wild-type (FY72), mcm4ts (FY364), Δcds1 (FY865), Δrad3 (FY1106), Δrad22 (FY1209), and Δrhp51 (FY1884) strains were grown in liquid cultures at the permissive temperature (25°C) in the presence of 15 mM HU. Aliquots of cells were plated to YES medium at the indicated time points and incubated at 25°C for 4 days; relative viability was calculated as the number of colonies at each time point relative to the number at the zero time point. (C) HR mutants generate fewer phospho-H2A damage foci than checkpoint mutants, suggesting that most replication forks are stabilized. Asynchronous cultures of wild-type (FY72), Δcds1 (FY865), Δrad3 (FY1106), Δrad22 (FY1209), and Δrhp51 (FY1884) cells grown at 25°C were treated with 15 mM HU for 4 h and then released into fresh medium without HU. At the indicated time points, spread nuclei were prepared and immunostained for phospho-H2A. Phospho-H2A focus formation in the Δrad3 mutant is due to activity of the Tel1 protein kinase, which acts redundantly with Rad3 (54, 83) (see Fig. S1 in the supplemental material). (D) Rad22-YFP foci form in cells recovering from HU-induced arrest. Asynchronous (asynch) cultures of wild-type (FY72) and Rad22-YFP (FY2878) cells were incubated with 20 mM HU for 4 h at 25°C. At 0, 2, and 4 h after the addition of HU, Rad22-YFP localization was examined and quantitated in live cells (FY2878). (E) Rhp51 localization was assayed by anti-Rhp51 immunostaining of spread nuclei prepared from wild-type cells (strain FY72) treated with HU for 0, 2, or 4 h. The percentage spread nuclei that contained Rhp51 nuclear foci is indicated in the text. DNA was counterstained with DAPI. Scale bar, 10 μM.