The minichromosome maintenance (MCM) complex plays essential conserved roles throughout DNA

The minichromosome maintenance (MCM) complex plays essential conserved roles throughout DNA synthesis: first as a component of the prereplication complex at origins and then like a helicase associated with replication forks. to cells of inactivating MCM function during S phase in the absence or the presence of forks stalled by HU. We demonstrate that the loss of MCM function produces DNA breaks cell cycle arrest and a loss of viability related to that observed in mutants that undergo replication fork collapse. Consistent with these results we find that Mcm4 interacts with the checkpoint protein kinase Cds1 and undergoes Cds1-dependent phosphorylation in cells treated with HU. This result suggests that MCM proteins take action to keep up replication fork structure both during normal S phase Crizotinib and during S-phase arrest. Our data additionally suggest that MCM function is Crizotinib required for appropriate recovery from replication arrest induced by HU. We observed an interaction between the MCM complex and the HR protein Rhp51 (Rad51). Although Rhp51 and additional HR proteins are not required to activate the replication checkpoint to keep up fork structure in HU or to restart DNA synthesis we find that the loss of HR function results in chromosome missegregation following launch from HU-induced replication arrest (this work; 48). We suggest that MCM proteins modulate replication fork progression arrest and restart and that they couple these functions with the restoration of DNA damage to guard S-phase genome stability. Our analysis in fission candida is definitely complemented by studies of mammalian cells suggesting that the part of the MCM complex in S-phase genome stability is conserved. MATERIALS AND METHODS Yeast strains and media. strains (Table ?(Table1)1) were constructed and maintained according to standard procedures (53). Cells were treated with 15 or RAD51A 20 mM HU (Sigma) for the indicated times or with 0.0025% methyl methanesulfate (Sigma) or 5 μU/ml bleomycin (Sigma) for 1 h or were irradiated with 100 Gy using a 60cobalt source. Asynchronous cultures or cultures synchronized by nitrogen starvation (53) were shifted to the restrictive temperature (36°C) for 4 h; cells pulse-labeled with bromodeoxyuridine (BrdU) were shifted to the restrictive temperature for 3.5 h and then incubated with 200 μg/ml Crizotinib BrdU (Sigma) for an additional 30 min at 36°C. TABLE 1. Yeast strains used in this study Cell culture and short interfering RNA (siRNA). HeLa cells were grown in Dulbecco’s modified Eagle’s medium containing 10% calf serum (Invitrogen) with appropriate antibiotics. HeLa cells were synchronized in G1/S with 2 mM thymidine (Sigma) for 18 h and then released into S phase for 3 to 4 4 h in the absence of added DNA damage. Cells were blocked in early S phase with 2.5 mM HU for 18 h. For siRNA experiments synthetic RNA duplexes were Crizotinib transfected into HeLa cells to a final concentration of 20 nM using Oligofectamine (Invitrogen) according to the protocol provided by Dharmacon. siRNA duplexes used were CGACAGCTAGAGTCATTAA for MCM4 and ATCGGATTGTGAAGATGAA for MCM7 synthesized by Integrated DNA Technologies Inc. Pools of four additional siRNA duplexes against MCM4 or MCM7 were obtained from Dharmacon. The siRNA negative control duplex was from Invitrogen. Relative viability Crizotinib was determined as the percentage of viable cells at 48 h compared to the percentage of viable cells during transfection (0 h). Immunofluorescence. Pass on nuclei were ready from fission candida cells relating to previous methods (29). Quickly cells had been spheroplasted in phosphate-buffered saline (PBS) with 0.5 mg/ml zymolyase 20T (Seikagaku) and 0.5 mg/ml lysing enzymes (Sigma). Cells had been then cleaned in MES [(2-chromatin materials were made by a modification of the protocol utilized to visualize candida artificial chromosome chromatin materials. The protocol generates materials of just one 1 approximately.8 kb/μm (65). Cells had been spheroplasted in zymolyase blend (1 M sorbitol 60 mM EDTA 100 mM sodium citrate pH 7 0.5 mg/ml zymolyase 20T 100 mM β-mercaptoethanol) and spheroplasts had been air dried onto Colorfrost slides. Materials were extended by pipetting 50 μl lysing remedy (50 mM Tris-Cl pH 7.4 25 mM EDTA 500 mM NaCl 0.1% [wt/vol] Nonidet P-40 1 [wt/vol] sodium dodecyl sulfate [SDS] 3 mM β-mercaptoethanol) onto the spheroplasts.