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Genes to Cells (2009) 14, 807-820. doi:10.1111/j.1365-2443.2009.01310.x
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Ctf4 coordinates the progression of helicase and DNA polymerase {alpha}

 Hirokazu Tanaka1,{dagger}, Yuki Katou1,{dagger}, Masaru Yagura2, Katsuya Saitoh1, Takehiko Itoh3, Hiroyuki Araki2, Masashige Bando1 and Katsuhiko Shirahige1

1 Laboratory of Chromosome Structure and Function, Department of Biological Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-20, 4259, Nagatsuta, Midori-ku, Yokohama City, Kanagawa 226-8501, Japan
2 Division of Microbial Genetics, National Institute of Genetics, Research Organization of Information and Systems, Sokendai, Yata 1111, Mishima, Shizuoka 411-8540, Japan
3 Research Center for Advanced Science and Technology, Mitsubishi Research Institute Inc., Chiyoda-ku, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Ctf4 is a protein conserved in eukaryotes and a constituent of the replisome progression complex. It also plays a role in the establishment of sister chromatid cohesion. In our current study, we demonstrate that the replication checkpoint is activated in the absence of Ctf4, and that the interaction between the MCM helicase-go ichi ni san (GINS) complex and DNA polymerase {alpha} (Pol {alpha})-primase is destabilized specifically in a ctf4{Delta} mutant. An in vitro interaction between GINS and DNA Pol {alpha} was also found to be mediated by Ctf4. The same interaction was not affected in the absence of the replication checkpoint mediators Tof1 or Mrc1. In ctf4{Delta} cells, DNA pol {alpha} became significantly unstable and was barely detectable at the replication forks in HU. In contrast, the quantities of helicase and DNA pol {varepsilon} bound to replication forks were almost unchanged but their localizations were widely and abnormally dispersed in the mutant cells compared with wild type. These results lead us to propose that Ctf4 is a key connector between DNA helicase and Pol {alpha} and is required for the coordinated progression of the replisome.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
During the S-phase of the eukaryotic cell cycle, the progression of DNA replication forks is carefully regulated to allow for the stable transmission of genetic information from generation to generation (Branzei & Foiani 2006). The replisome is a macromolecular complex consisting of essential replication proteins including DNA helicase and DNA polymerases, as well as additional non-essential factors that facilitate faithful replication of chromosomal DNA (Branzei & Foiani 2006). The biochemical identification of a subcomplex of the replisome, the replisome progression complex (RPC), showed that it consists of the minichromosome maintenance (MCM) proteins, the likely replicative DNA helicase, the go ichi ni san (GINS) complex, Cdc45, Mrc1, Tof1, Csm3, facilitates chromatin transcription (FACT) and Ctf4 (Gambus et al. 2006). Interestingly, no DNA polymerases co-purified with RPC. Thus, the mode of association of the RPC with DNA polymerases during the synthesis of leading and lagging strands is not clear at present.

The GINS complex consists of the Psf1, Psf2, Psf3 and Sld5 proteins. GINS is required for both the initiation of chromosome replication and the normal progression of DNA replication forks during S-phase (Kanemaki et al. 2003; Takayama et al. 2003). GINS localizes at the replication forks with the Mcm2–7 proteins that are suggested to form the catalytic core of the essential replicative helicase (Ishimi 1997; Kanemaki & Labib 2006; Moyer et al. 2006; Pacek et al. 2006). The Cdc45 protein forms a tight complex with MCM at DNA replication forks and is also required for unwinding of the DNA duplex to allow progression of the forks (Aparicio et al. 1997; Labib et al. 2000). Mrc1, Tof1 and Csm3 are not essential under unchallenged conditions, but are required for activation of the DNA replication checkpoint in response to replication fork stalling after treatment with the ribonucleotide reductase inhibitor HU (Alcasabas et al. 2001; Foss 2001; Katou et al. 2003; Tourrière et al. 2005). Ctf4 was initially identified as a polymerase {alpha} (Pol {alpha}) associated protein (Miles & Formosa 1992) and is required for cohesion establishment during S-phase (Lengronne et al. 2006). The histone chaperone FACT (Spt16 and Pob3) is considered to be required for transcription and replication to proceed through chromatin (Schlesinger & Formosa 2000; Tan et al. 2006).

Studies on Ctf4 thus far have suggested that it connects proteins involved in DNA replication, the replication checkpoint, chromatin remodeling and chromosomal partition (Ho et al. 2002; Warren et al. 2004; Pan et al. 2006; Collins et al. 2007). Deletion of CTF4 leads to a delay in cell cycle progression through mitosis, as well as a sister chromatid cohesion defect (Hanna et al. 2001). Similar phenotypes to those of the budding yeast ctf4 deletion mutant were observed in fission yeast cells lacking the CTF4 orthologue MCL1 (Williams & McIntosh 2002). Mcl1 interacts physically with Pol {alpha} and is associated with chromatin in G1 and S-phase, but not in G2, suggesting a function for this protein during S-phase (Williams & McIntosh 2002, 2005; Tsutsui et al. 2005). Cells lacking Mcl1 exhibit defects in sister chromatid cohesion and chromosome segregation. Recent reports have suggested that Mcl1 is involved in heterochromatin formation specifically in the outer centromeric regions. Deletion of mcl1 have also been shown to lead to a loss of Cnp1-GFP (a CENP-A homologue in Schizosaccharomyces pombe) foci from the nucleus (Mamnun et al. 2006), suggesting that Mcl1 functions during kinetochore assembly. And-1, a homologue of Ctf4 in Xenopus, was also suggested to be required for loading of the Pol {alpha} complex at the initiation step of DNA replication (Zhu et al. 2007).

The aim of our current study was to further elucidate the molecular functions of Ctf4 during chromosomal replication. The results of our in vivo and in vitro analyses using a ctf4 deletion mutant suggest that Ctf4 is a key connector of the GINS-MCM helicase complex and Pol {alpha}. In ctf4{Delta} cells, Pol {alpha} and RPA were barely detectable and both helicase and DNA pol {varepsilon} showed a widely dispersed localization compared with wild type in HU. These results lead us to propose Ctf4 is a key connector between the DNA helicase and Pol {alpha} and is required for the coordinated progression of these two enzymes. In addition, cohesion defects in ctf4{Delta} cells may be the result of an impaired DNA replication fork structure, unsuitable for proceeding through cohesin rings (Lengronne et al. 2006).


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
CTF4 deletion leads to low level of replication checkpoint activation

Ctf4 is a constituent of the replication machinery (Gambus et al. 2006; Lengronne et al. 2006) and the corresponding deletion mutation causes synthetic growth defects when combined with a loss of the DNA replication checkpoint genes, MRC1, TOF1 and CSM3 (Warren et al. 2004; Collins et al. 2007). Based on these reports, we herein further examined the role of Ctf4 in S-phase progression. We analyzed the cell-cycle distribution of ctf4{Delta} cells during asynchronous growth in YPD medium (Fig. 1A). The data showed that in the absence of CTF4, the cells accumulate at the S-G2 boundary of the cell-cycle. This delay was previously reported to be partially due to the activation of a spindle checkpoint (Hanna et al. 2001). Since the accumulation of cells in S-G2 may also be the consequence of a DNA damage checkpoint (Weinert 1998), we deleted rad9, a mediator of such checkpoints, in the ctf4{Delta} background (Fig. 1A). The cell-cycle distribution of the resulting double deletion mutant became similar to wild type, suggesting that the accumulation of S-G2 cells in the ctf4 deletion mutant was indeed partly due to the activation of a DNA damage checkpoint.


Figure 1
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  		Figure 1  A rad9-dependent DNA damage checkpoint is activated in the ctf4{Delta} mutant. (A) Cell-cycle distribution of wild-type, and ctf4{Delta}, rad9{Delta}, and ctf4{Delta}rad9{Delta} mutants. DNA content was analyzed by flow cytometry. Cells were synchronized in G1-phase with {alpha}-factor and then released into S-phase at 23 °C. Samples were withdrawn at the indicated times. (B) Extracts were prepared from the same samples described in panel (A), and analyzed by Western blotting using a specific antibody against Rad53. A phosphorylated form of Rad53 is indicated by the arrowhead.

 
To address this point further, we analyzed the phosphorylation status of Rad53, an effector kinase of the DNA damage/replication checkpoint, during synchronous S-phase progression as this protein is known to be phosphorylated in response to DNA damage or stalled replication (Zhou & Elledge 2000). Cells (ctf4{Delta}, rad9{Delta}, ctf4{Delta}rad9{Delta} and wild type) were arrested by {alpha}-factor and then released into S-phase. The cell-cycle distribution and Rad53 phosphorylation patterns were analyzed by FACS and Western blotting, respectively, every 20 min after the release (Fig. 1A and B). The cell-cycle progression of the ctf4 deletion mutant was delayed at late S-G2 phase as compared with wild type and the rad9 deletion mutant, and this delay was partly suppressed in the double deletion mutant (Fig. 1A). In the ctf4 deletion mutant, Rad53 was found to be already phosphorylated in asynchronous cultures (Fig. 1B). In cultures with a synchronized cell-cycle, Rad53 phosphorylation reached higher levels, and persisted until later timepoints in the ctf4{Delta} cells. In wild-type cells, significant Rad53 phosphorylation was not observed. Rad53 phosphorylation was reduced in the ctf4{Delta}rad9{Delta} cells to the same levels seen in the rad9 single deletion mutant and cell cycle progression through G2 was closer to wild type as compared the ctf4 single deletion mutant. We conclude from these data that DNA damage occurs during S-phase in the absence of Ctf4, leading to activation of the Rad9-dependent DNA damage response.

Ctf4 is required for the coordinated progression of DNA replication forks

To examine the possible role of Ctf4 in the DNA replication checkpoint, we examined the HU sensitivity of the ctf4 deletion mutant (Fig. 2A). Cells lacking Ctf4 were found to be sensitive to HU, and this sensitivity was further increased by the deletion of rad9. As this phenotype was similar to that of deletion mutants for tof1 (Foss 2001), a gene which functions upstream of the DNA replication checkpoint, we analyzed whether the same was true for ctf4 mutants. For this purpose, we analyzed the Rad53 phosphorylation status of both wild-type yeast and ctf4 deletion mutants in the presence of HU (Fig. 2B). We also examined the ctf4{Delta}rad9{Delta} double deletion mutant as the Rad9-dependent DNA damage checkpoint phosphorylates Rad53 redundantly with the DNA replication checkpoint pathway in response to fork stalling (Alcasabas et al. 2001). As a control, we included a tof1{Delta}rad9{Delta} double deletion mutant in which Rad53 phosphorylation is reduced. In contrast, Rad53 became phosphorylated in response to HU in the ctf4{Delta}rad9{Delta} mutant, suggesting that the activation of the DNA replication checkpoint does not require Ctf4.


Figure 2
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  		Figure 2  The ctf4{Delta} mutant can activate the DNA replication checkpoint normally in response to HU. (A) Cells were grown in YPD at 23 °C. Serial 10-fold dilutions of these cultures were then spotted onto YPD or HU-containing YPD plates and incubated at 30 °C for 3 days. Wild-type yeast, and the mutants ctf4{Delta}, rad9{Delta} and ctf4{Delta}rad9{Delta} were used. (B) Rad53 phosphorylation was induced normally in response to replicative stress by HU in the ctf4{Delta} mutant. Cells were arrested in G1-phase by {alpha}-factor and then released into S-phase at 23 °C in the presence of 200 mM HU. The samples were withdrawn at the indicated times and cell extracts were prepared. Rad53 was detected by Western blotting. A phosphorylated form of Rad53 is indicated by a bracket. (C) Distribution of Cdc45 on yeast chromosome VI in a wild-type strain, tof1{Delta} mutant strain and the ctf4{Delta} mutant strain in the presence of 200 mM HU. Cells were arrested in G1-phase by {alpha}-factor and then released into S-phase at 23 °C in the presence of 200 mM HU for 60 min. (D) Efficiency of chromatin immunoprecipitation (ratio of input vs. IP) of the Cdc45-3xHA-chromatin complex was measured by real-time PCR using primer pairs that specifically amplify the regions indicated by the arrowheads in (C). The results are an average of two independent experiments with standard deviations. 197 and 259K indicate the positions of the PCR primers in kilobases from the left end of the chromosome.

 
Under the same experimental conditions, we determined the binding profile of Cdc45, a protein associated with the MCM helicase complex, and tested for DNA synthesis on chromosome VI via a BrdU incorporation assay using the ChIP-chip technique in wild-type and the ctf4{Delta} mutant (Fig. 2C). In wild-type cells, only early firing origins were found to be activated in the presence of HU (ARS 603.5, 605, 606 and 607 on chromosome VI), while late firing origins (ARS 603, 608 and 609) remained silent. However, late origins have been shown to be activated in the presence of HU when the DNA replication checkpoint is defective (Santocanale & Diffley 1998). As expected in our present analysis, only early firing origins (ARS603.5, 605, 606 and 607) were activated in wild-type cells, whereas the late firing origin ARS608 was activated in tof1{Delta} cells. In the ctf4{Delta} mutant, no activation of late firing origins occurred, suggesting that the DNA replication checkpoint is functional in these cells.

During our analysis of replication origin firing, we noticed that the binding of Cdc45 and the incorporation of BrdU into active origins was quite widely dispersed in the ctf4{Delta} mutant, suggesting that the replisome had stalled asynchronously. We further analyzed the levels of Cdc45 associated with replication forks by measuring the amount of DNA associated with Cdc45 by quantitative PCR (q-PCR) (Fig. 2D). We analyzed a region at 197 kb from the left telomere and next to the early firing origin ARS607, where Cdc45 is well-enriched, and also a control locus close to ARS609 that remains inactive after HU treatment. The amount of Cdc45 associated with the replication forks at this 197 kb position was reduced by half in the ctf4{Delta} mutant as compared with the wild-type control, in good agreement with our ChIP-chip data (Fig. 2C and D). However, the region around ARS607 bound by Cdc45 in ctf4{Delta} was increased twofold compared with wild-type cells (Fig. 2C), suggesting that level of Cdc45 associated around this origin is unaffected in ctf4{Delta} mutant.

We tested the possibility that the replisome became asynchronously arrested in the ctf4{Delta} mutant in response to the induction of replicative stress by HU by examining the association of additional fork components at chromosome VI (Fig. 3). We performed ChIP-chip and ChIP-qPCR analyses for Pol {alpha}, Mcm4 and Dpb2 (Fig. 3A and B) in HU-treated yeast strains. The levels of Pol {alpha}, Mcm4 and Dpb2 found to be associated with the replication forks at the 197 kb region on chromosome VI were reduced to 24%, 35% and 50%, respectively, in ctf4{Delta} compared with wild-type control (Fig. 3B). As these reduction ratios were in good agreement with our ChIP-chip data (Fig. 3A), we estimated the amount of these proteins that had bound the region around the ARS607 in the ctf4{Delta} mutant by measuring the blue colored area and compared this value with the corresponding measurement in wild-type cells (Fig. 3C). The data show that the most significant reduction (to about half of the wild-type levels) was observed for Pol {alpha} (45%), but that more than 80% of the Cdc45, Mcm4 and Dpb2 proteins were still associated with the replication forks around ARS607 in ctf4{Delta}. These data suggest that the loss of Ctf4 affects the stable association of the DNA pol {alpha} complex to the forks and thereby leads to asynchronous arrest of the helicase complex as well as the leading strand polymerase. We further analyzed the distribution of the single stranded DNA binding protein RPA by ChIP-chip and found that its localization was also widely dispersed in the ctf4{Delta} mutant (Fig. 3D). This RPA distribution may not indicate that extensive damage has occurred due to the absence of Ctf4, as we could not detect enhanced recruitment of Ddc2 and Ddc1, checkpoint sensor proteins, to replicating regions (data not shown). The distribution of RPA more closely reflected a lagging strand synthesis complex localization pattern because RPA is a component of the DNA replication fork and preferentially binds to the lagging strand.


Figure 3
Figure 3
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  		Figure 3  Localization of the replication proteins in the ctf4{Delta} mutant. (A) Distribution of Pol {alpha}, Mcm4 and Dpb2 on chromosome VI in wild-type and ctf4{Delta} strains in the presence of 200 mM HU. Cells were arrested in G1-phase by {alpha}-factor and then released into S-phase at 23 °C in the presence of 200 mM HU for 60 min. Pol {alpha} and Dpb2 were tagged by 3xFLAG at their C-termini. Mcm4 was tagged by 3xHA at its C-terminus. (B) Efficiencies of chromatin immunoprecipitation (ratio of input vs. IP) with Pol {alpha}-3xFLAG, Mcm4-3xHA, and Dpb2-3XFLAG were measured by real-time PCR using primer pairs that specifically amplify the regions pointed by arrowheads in (A) and are denoted by the distances in kilobases from the left end of the chromosome. The results are an average of two independent experiments with standard deviations. (C) Ratios (wild type vs. ctf4{Delta}) of fork bound Pol {alpha}-3xFLAG, Mcm4-3xHA, and Dpb2-3XFLAG were estimated by measuring the blue colored area around ARS607 of ChIP-chip data (Figs 2C and 3A). The blue colored area of 180–215kb was measured for Cdc45. For the other three proteins, 190–210kb was measured. (D) Distribution of Rfa1, the largest subunit of RPA, on chromosome VI in wild-type and ctf4{Delta} yeast in the presence of 200 mM HU. Cells were arrested in G1-phase by {alpha}-factor and then released into S-phase at 23 °C in the presence of 200 mM HU for 60 min. Rfa1 was tagged by 3xFLAG at its C-terminus. (E) Distribution of Pol {alpha} and Cdc45 on chromosome VI in wild-type and ctf4{Delta} yeast during normal S-phase progression. Cells expressing a tagged version of each protein were arrested in G1 by {alpha}-factor and then released into S phase for 45 min by addition of pronase at 16 °C to slow down DNA synthesis. Pol {alpha} and Cdc45 were tagged by 3XFLAG and 3XHA, respectively.

 
To confirm that unstable association of DNA pol {alpha} and asynchronous progression of helicase were not the consequence of replicative stress by HU, protein-binding profiles during early replication in the absence of HU were determined. Cells were released from G1 arrest at low temperature (16 °C) in the medium without HU to slow the DNA synthesis, and locations of Cdc45 and Pol {alpha} were analyzed (Fig. 3E) after 45 min of release as previously described (Katou et al. 2003). The data show that Pol {alpha} binding was hardly detectable. In contrast, Cdc45 was still associated with the replication forks but widely dispersed just like in HU. These data strongly suggest that the loss of Ctf4 affects the stable association of the DNA pol {alpha} complex to the forks and leads to asynchronous progression of the helicase complex in normal S-phase.

Ctf4 is required for the stable interaction between Pol {alpha} and the RPC

Since Ctf4 is known to be associated with Pol {alpha} (Miles & Formosa 1992), and forms part of the RPC (Gambus et al. 2006), we examined its role in the interaction between these two protein complexes. First, we examined the interaction between these two complexes by co-immunoprecipitation of Pol {alpha} and Cdc45 in S-phase cells, either after synchronous release from a {alpha}-factor block in G1, or after arrest by HU treatment (Fig. 4A). Our results clearly demonstrated an interaction between Pol {alpha} and Cdc45 occurs specifically in S-phase. In the absence of Ctf4, the association of Cdc45 with Pol {alpha} was essentially abolished. The same Ctf4-dependence was observed for the interactions between Tof1 and Pol {alpha}, the Pol {alpha} primase subunit Pri1 and Mcm2, and also the GINS subunit Psf1 and Pol {alpha} (Fig. 4B–D). The data demonstrate that Ctf4 is indeed required to mediate interactions between Pol {alpha} and the RPC. The integrity of the RPC itself, or the Pol {alpha}-primase complex, was not compromised by the absence of Ctf4, as the interactions between Pri1 and Pol {alpha}, Psf1 and Mcm2, Cdc45 and Mcm2, and Tof1 and Mcm2 were unaffected (Fig. 4C–F). In addition, the interaction between Cdc45 and Dpb2, a subunit of the leading strand DNA pol {varepsilon}, was slightly reduced in the ctf4{Delta} mutant (Fig. 4G), suggesting that Ctf4 may also have a minor role in the stabilization of leading strand polymerase. Interestingly, the replication checkpoint proteins, Tof1 and Mrc1, that are required for the stable arrest of the replication forks in response to HU, had no effect on the interactions between Pol {alpha} and Mcm2, Pol {alpha} and Cdc45, and Psf1 and Pol {alpha} (Fig. 4H–K), suggesting that Ctf4 has a specific role in the interaction between the Pol {alpha}-primase complex and the RPC and is important for the maintenance of replisome integrity after fork arrest by HU.


Figure 4
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  		Figure 4  Ctf4 is required for a stable interaction between the Pol {alpha}-primase complex and various components of the RPC. (A) Interaction between Pol {alpha} and Cdc45. Immunoprecipitations with anti-FLAG (FLAG IP) antibody using extracts of wild-type and ctf4{Delta} cells co-expressing Pol {alpha}-3xFLAG and Cdc45-3xHA are shown. Cells were grown exponentially (Asyn.), synchronized in G1 by {alpha}-factor (G1), and then either released from G1 into S-phase (S), or released from G1 into S-phase in the presence of 200 mM HU (HU) for 60 min. Pol {alpha} and Cdc45 were then detected by Western blotting analysis of immunoprecipitates (IP) and of whole cell extracts (WCE) as a control using anti-FLAG (top) or anti-HA (bottom) antibodies. The same procedures were used to analyze interactions between Pol{alpha} and Tof1 (B); between Pri1 and Pol {alpha} and between Pri1 and Mcm2 (C); the effects of Tof1 and Ctf4 on the interaction between Psf1, Mcm2, and Pol {alpha} (D); interaction between Cdc45 and Mcm2 (E); interaction between Tof1 and Mcm2 (F); interaction between Cdc45 and Dpb2 (G); the effects of Tof1 and Ctf4 on the interaction between Pol {alpha} and Mcm2 (H); effects of Tof1 on the interaction between Pol {alpha} and Cdc45 (I); effects of Mrc1 on the interaction among Pol {alpha}, Cdc45, and Mcm2 (J); and the effects of Mrc1 on the interaction among Psf1, Mcm2, and Pol {alpha} (K).

 
Ctf4 links the interaction between GINS and Pol {alpha} in vitro

As Ctf4 is a component of the RPC, a biochemically identified complex of the replisome during S-phase progression (Gambus et al. 2006), we wanted to identify interacting partners of Ctf4 within this important complex. We thus overexpressed a GST fusion protein product of each subunit of the RPC together with a HA epitope tagged Ctf4 under the control of the GAL1 promoter, and examined their interactions by co-immunoprecipitation analyses (Fig. 5A–C). When we used RPC subunits as baits, strong interaction between Ctf4 and Sld5 and weak interaction between Psf3 and Ctf4 were detected (Fig. 5A and B). The results revealed that Ctf4 specifically interacts with Sld5, both when Ctf4 was used as the bait and reciprocally, (Fig. 5C), but not with any other component of the RPC. This finding suggests that Ctf4 directly interacts with GINS through Sld5 to connect Pol {alpha} with the GINS complex. To further evaluate this possibility, we examined whether Ctf4 is required for the interaction between GINS and Pol {alpha} in vitro (Fig. 5D and E). We purified GINS from Escherichia coli cells co-expressing Psf1, 2, 3 and Sld5, and incubated the complex with Pol {alpha} purified from yeast cells with or without Ctf4. The obtained data clearly showed that the GINS complex and DNA Pol {alpha} interact only when Ctf4 is present (Fig. 5E). This suggests that Ctf4 mediates an interaction between the Sld5 subunit of the GINS complex and Pol {alpha} within the eukaryotic replisome.


Figure 5
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  		Figure 5  Ctf4 interacts directly with Sld5 and connects GINS and DNA Pol {alpha} in vitro. (A) An anti-GST IP was carried out on strains co-overexpressing Ctf4-3xHA and each subunit of GINS fused to GST. Each member of the RPC was fused to the GAL1-10 promoter, cloned into the multi-copy plasmid pEG(KT), and co-overexpressed with HA tagged Ctf4 in yeast strains for 3 h at 23 °C by adding galactose to a final concentration of 2%. The soluble protein extracts were then incubated with glutathione-Sepharose. The proteins retained on the sepharose were analyzed by SDS-PAGE and subsequent Western blotting using anti-GST, and anti-HA antibodies. Lane 1, GST only; lane 2, GST-Sld5; lane3, GST-Psf1; lane 4, GST-Psf2; lane 5, GST-Psf3. Blots of the IPs were probed with anti-GST (top) or anti-HA (bottom) antibody. (B) An anti-GST IP was carried out on strains co-over-expressing Ctf4-3xHA and each member of the RPC fused to GST. Lanes 1, GST-Mcm2; lane 2, GST-Mcm3; lane3, GST-Mcm4; lane 4, GST-Mcm5; lane 5, GST-Mcm6; lane 5, GST-Mcm6; lane 6, GST-Mcm7; lane 7, GST-Mcm10; lane 8, GST-Cdc45; lane 9, GST-Sld5; lane 10, GST only. Blots of the IPs were probed with anti-GST (top) or anti-HA (bottom) antibody. (C) An anti-HA IP was carried out on strains overexpressing each subunit of GINS fused to GST with or without overexpressed Ctf4-3xHA. Lanes 1–4, overexpression of the indicated subunit of GINS. Lanes 5–8 co-overexpression of the indicated subunit of GINS and Ctf4. Blots of the IPs were probed with anti-HA (top) or anti-GST (bottom) antibody. (D) SDS-PAGE analysis of purified recombinant proteins used for in vitro interaction study described in (E). The purified proteins were separated by 5%–20% SDS-PAGE and subsequently silver-stained. (Left panel) GST (lane 1), GST-Ctf4 (lane 2) and GINS (Sld5, Psf1, Psf2 and 6His-Psf3; lane3). (Right panel) lane 4 contains the Pol {alpha} complex (Pol {alpha}, Pol12-CBP, Pri2 and Pri1). (E) Ctf4 is required for the interaction between GINS and Pol {alpha} complex in vitro. The Pol {alpha} complex was purified from yeast expressing Pol12-TAP using tandem affinity purification. GINS was purified from an E. coli strain co-expressing all four subunits of GINS. GST-Ctf4 was purified from yeast over-expressing GST-Ctf4. Purified GST (1 pmol) or GST-Ctf4 (0, 0.25, 0.5 and 1 pmol) was mixed with GINS (1 pmol) and CBP-tagged Pol {alpha} (1 pmol) pre-bound to Dynabeads M270 Epoxy. Proteins bound to the beads were resolved by SDS-PAGE and analyzed by Western blotting using anti-GST, anti-CBP and anti-Sld5 antibodies.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In our current study, we analyzed the function of Ctf4, a well-conserved protein in eukaryote species, at a molecular level. Several non-essential proteins have been so far reported to interact with replication forks including Mrc1, Tof1, Csm3 and Ctf4, among which Mrc1, Tof1 and Csm3 were characterized as replication checkpoint proteins that function in the stable pausing of replication forks and checkpoint activation (Katou et al. 2003; Calzada et al. 2005; Szyjka et al. 2005; Tourrière et al. 2005). Ctf4 was initially identified as a Pol {alpha} interacting factor and was also reported to be required for cohesion establishment (Miles & Formosa 1992; Hanna et al. 2001). We found in our current analyses that Ctf4 is also required for the interaction between Pol {alpha}-primase complex and RPC, a helicase containing replisome subcomplex. We further found that Ctf4 directly interacts with Sld5, a subunit of GINS and is also required for the interaction between DNA Pol {alpha} and GINS in vitro. Moreover, the interaction between RPC and DNA Pol {alpha} complex is dependent on Ctf4, but not on other DNA replication checkpoint proteins like Tof1 or Mrc1. In contrast, the deletion of Ctf4 did not cause any defects in checkpoint activation even when combined with a rad9 deletion unlike the checkpoint signaling defects caused by the loss of other non-essential components such as Tof1, Csm3 and Mrc1.

Interestingly, the deletion of Ctf4 caused a reduction in binding of DNA Pol {alpha} in the presence of HU. The levels of binding of other essential components at the replication forks including Cdc45, Mcm4 or Dpb2, were not severely affected in the ctf4{Delta} mutant but their distribution was widely dispersed compared with wild-type cells exposed to HU. The loss of Pol {alpha} binding and widely dispersed localization of Cdc45 were observed during normal progression of S-phase in ctf4{Delta} mutant. Based on these results, we propose that Ctf4 plays a role in the coordinated progression of helicase, and of leading, and lagging-strand synthesis during DNA replication. It is interesting to note in this regard that the interaction between DNA Pol {alpha} and the helicase complex is not affected by the deletion of replication checkpoint mediator proteins such as Tof1 or Mrc1, which are known to have functions in the coupling of replicated regions and the replication machinery (Katou et al. 2003). Tof1 and Mrc1 may thus mainly function in the maintenance and stabilization of DNA-replisome interactions rather than as components of the supporting structure of the replisome.

The uncoupling of Pol {alpha} and GINS was found not to be lethal and only caused slight damage during the normal process of DNA replication. Pol {alpha} is known to interact with Mcm10 (Zhu et al. 2007), which may help with the recruitment and maintenance of Pol {alpha} at the replication fork in the ctf4{Delta} mutant. Pol {alpha} can be bound to the template by its own ssDNA binding activity in the absence of Ctf4 to support DNA replication. Recent reports by Zhu et al. have suggested that And-1, a homologue of Ctf4 in Xenopus, is required for the loading of the Pol {alpha}-primase complex at the initiation step of DNA replication (Zhu et al. 2007). However, it is not yet clear whether And-1 is also required during the progression of DNA replication forks in the Xenopus system. Hence, although it is currently difficult to identify the exact step at which Ctf4 plays the essential role in S-phase, our previous analyses of Ctf4 localization at replication forks during DNA replication (Lengronne et al. 2006) argue that it functions during DNA replication as a coupler of DNA helicase and the lagging strand DNA polymerase.

Ctf4 is known as a factor important for cohesion establishment among a group of proteins that includes Tof1, Csm3, Mrc1, Rrm3, Ctf18, Pol {varepsilon} and Chl1. Many of these cohesion establishment factors are involved in the process of DNA replication or DNA replication checkpoint (Edwards et al. 2003; Mayer et al. 2004; Petronczki et al. 2004; Skibbens 2004; Warren et al. 2004). Based on these results, we propose that an irregular fork structure in the ctf4{Delta} mutant would fail to pass through local chromatin regions bound with cohesin and therefore may inhibit cohesion formation between sister chromatids during DNA replication. Recently it was reported that for cohesion establishment, acetylation of Smc3, a cohesin subunit, by Eco1 is essential. It would therefore be interesting to analyze the acetylation of Smc3 in the ctf4{Delta} mutant in a future study to examine whether Ctf4 has a direct role in the acetylation process.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Yeast strains

All yeast strains used in this study are listed in Table 1 and are derivatives of BY4741 (MATa, his3{Delta} 1 leu2{Delta} 0 met15{Delta} 0 ura3{Delta} 0). In all cases, a 3xHA or 3xFLAG tag was fused to the C terminus of the protein of interest. A 6xHis + 3xHA or 6xHis + 3xFLAG tag was fused to the C terminus of the protein of interest using a cassette amplified from pU6H3HA or pU6H3FLAG as previously described (Katou et al. 2003). All strains expressing a tagged protein were checked for unaltered doubling time and sensitivity to HU and MMS (methyl methane sulfonate) to ascertain the normalcy of the cells. Gene deletions were performed by replacing each open reading frame from the start to stop codon with a selective marker. The selective markers were amplified from YIplac128, YIplac204 and YIplac211 as described previously (Katou et al. 2003). For the construction of the strain that overexpresses Ctf4, the galactose inducible promoter (GAL1 promoter) with a selectable marker was amplified from pFA6a-HisMX6-PGAL1-3HA and used to replace the native CTF4 promoter (Longtine et al. 1998). All plasmids used in this study are listed in Table 2.


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  		Table 1  Yeast strains used in this study
 

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  		Table 2  Plasmids used in this study
 
Antibodies

The HA and FLAG epitopes were detected using the mouse monoclonal antibodies 12CA5 (final concentration 1 µg/mL) and M2 (final concentration 1 µg/mL), respectively. The endogenous Mcm2 and Rad53 proteins were detected by the goat polyclonal antibodies, sc-6680 (final concentration 0.2 µg/mL; Santa Cruz Biotechnology) and sc-6749 (final concentration 0.2 µg/mL; Santa Cruz Biotechnology), respectively.

GST pull down assays

For galactose-dependent induction and GST fusion, the coding region of each member of the RPC was amplified by PCR and cloned into pEG(KT) (Lei et al. 1996). Each member of the RPC was co-overexpressed with HA tagged Ctf4 in yeast strains for 3 h at 23 °C by adding galactose to a final concentration of 2%. Harvested yeast cells were homogenized using glass beads in lysis buffer (50 mM Hepes–KOH pH 7.5, 150 mM NaCl, 3 mM MgCl2, 10% glycerol, 0.2% NP-40 1 mM dithiothreitol) containing protease inhibitors (1x Complete (Roche), 1% Protease Inhibitor Cocktail (Sigma-Aldrich) and phosphatase inhibitors (10 mM NaF, 20 mM Glycerol 2-phosphate disodium salt hydrate). The soluble protein extracts were incubated with glutathione-sepharose 4B (GE Healthcare) in suspension at 4 °C for 3 h. The resin was washed extensively with lysis buffer containing 0.3 M NaCl and then boiled with SDS sample buffer at 95 °C for 3 min.

GINS-Ctf4-Pol {alpha} pull down assay

Anti-TAP antibodies (Open Biosystems) were mixed with 1.5 x 107 dynabeads M-270 epoxy (Dynal) in 12 µL Buffer A (0.1 M potassium phosphate, 1.5 M ammonium sulfate) at 37 °C for 30 h and then washed with 15 µL 0.15 M potassium acetate three times. The anti-TAP antibodies bound Dynabeads were then mixed with 0.1 M ethanolamine in 12 µL 0.15 M potassium acetate, incubated at 4 °C for 2 h, and washed twice with 15 µL 0.15 M potassium acetate. The beads were subsequently mixed with 1 pmol Pol {alpha} in 50 µL Buffer B (50 mM Hepes–KOH pH 7.5, 150 mM potassium acetate, 2 mM magnesium acetate, 20 µM ZnSO4, 10% glycerol, 0.1% Tween20, 0.01% nonidet P-40), incubated at 4 °C for 1 h, and then washed with 500 µL Buffer B. The resultant Pol {alpha}-bound Dynabeads were mixed with 1 pmol GINS and 1 pmol GST or 0, 0.25, 0.5 and 1 pmol GST-Ctf4 in 50 µL Buffer B containing 1 mg/mL BSA, incubated at 4 °C for 1 h, and then washed with 500 µL Buffer B. The proteins retained on the beads were analyzed by SDS-PAGE and subsequent Western blotting using anti-GST, anti-CBP and anti-Sld5 antibodies. Visualization of each antibody was performed with an Odyssey infrared imaging system (LI-COR).

ChIP-chip analyses

Chromatin immunoprecipitation on a DNA chip (ChIP-chip) was performed as previously described (Katou et al. 2003). To examine the whole region of chromosome VI, the RikDacF (Affymetrix) DNA chip was used. We disrupted 1.5 x 108 cells using a Multi-Beads Shocker (MB400U, Yasui Kikai). ChIP DNA was purified and amplified by random priming. Hybridization, washing and array scanning were carried out using the Affymetrix GeneChip system, according to the manufacturer's instructions. To discriminate positive and negative binding signals, we used three criteria: the reliability of the signal strength was judged by the P-value of each locus (P = 0.025); the reliability of the binding ratio was judged by a change in the P-value (P = 0.025); and clusters consisting of at least three contiguous loci which satisfy these first two criteria in two independent experiments were selected because a single protein–DNA interaction site will result in immunoprecipitation of DNA fragments that hybridize not only to the locus of the actual binding site but also to its neighbors. BrdU incorporation analyses were performed as previously described (Katou et al. 2003).

ChIP-Real-time PCR analysis

Real-time PCR was performed using the Applied Biosystems 7500 real-time PCR system according to manufacturer's instructions. The primer pairs 185 and 196 kb span the following positions on yeast chromosome VI, respectively: 184962–185178 (5'-CCTCC AAATCCACTGTTACTGCTATCC-3' and 5'-CCATTGTT ACTGGTCTTGTCCTTCTTGC-3'); 196898–197077, (5'-TGTAATACTCCTCAAAGGTCCTCC-3' and 5'-GAAGAC AACGACGAAGAACTAGC-3').

Co-immunoprecipitation analyses

The cells were harvested and washed two times with water supplemented with 0.1 mM phenylmethanesulfonyl fluoride (PMSF) as previously described (Katou et al. 2003). The cells were frozen as pellets by dropping into liquid nitrogen. The frozen cell pellets were broken in dry ice powder using a grinder. The homogenate was resuspended in 600 µL of lysis buffer (50 mM Hepes–KOH pH 7.5, 150 mM NaCl, 3 mM MgCl2, 10% Glycerol, 0.2% NP-40) containing protease inhibitors [1x Complete (Roche), 1% Protease Inhibitor Cocktail (Sigma)] and phosphatase inhibitors (10 mM NaF, 20 mM Glycerol 2-phosphate disodium salt hydrate). To analyze the Psf1-6xFLAG and Pol {alpha}-3xHA interaction, lysis buffer was changed to lysis buffer B (50 mM Hepes–KOH pH 7.5, 100 mM potassium acetate, 5 mM Magnesium acetate, 10% Glycerol, 0.1% NP-40) containing protease inhibitors [1x Complete (Roche), 1% Protease Inhibitor Cocktail (Sigma)] and phosphatase inhibitors (5 mM NaF, 10 mM Glycerol 2-phosphate disodium salt hydrate). The lysate were treated with 200 units of DNase I (TAKARA BIO) at 4 °C for 15 min. and then centrifuged to prepare a cleared whole-cell extract. For pre-clear, the lysate was incubated with either 20 µL of Sepharose beads (SIGMA) or Dynabeads Protein A for 30 min. After pre-clear, lysate was incubated with either ANTI-FLAG M2 Affinity Gel (SIGMA) or ANTI-HA (12CA5; Roche) conjugated with DynaBeads Protein A for 3 h. After immune-precipitation, samples were washed four times with lysis buffer, and then resuspended in SDS loading buffer (62.5 mM Tris–HCl pH 6.8, 2% sodium lauryl sulfate, 10% glycerol, 5% 2-mercaptethanol).


   Acknowledgements
 
We thank Dr Hiroshi Yoshikawa for critical reading of the manuscript. We also thank K. Kurihara and K. Nakagawa for their technical assistance; K. Hirai, Y. Li and T. Umemori for preparing purified proteins. We also thank all members of the Shirahige laboratory. K.S. and H.A. are supported in part by grants-in-aid on priority areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Y.K. is a GCOE Research Associate.


   Footnotes
 
Communicated by: Hiroshi Handa

{dagger}These two authors contributed equally to this work. Back

* Correspondence: mbando{at}bio.titech.ac.jp or kshirahi{at}bio.titech.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Alcasabas, A.A., Osborn, A.J., Bachant, J., Hu, F., Werler, P.J., Bousset, K., Furuya, K., Diffley, J.F., Carr, A.M. & Elledge, S.J. (2001) Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat. Cell Biol. 3, 958–965.[CrossRef][Medline]

Aparicio, O.M., Weinstein, D.M. & Bell, S.P. (1997) Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91, 59–69.[CrossRef][Medline]

Branzei, D. & Foiani, M. (2006) The Rad53 signal transduction pathway: Replication fork stabilization, DNA repair, and adaptation. Exp. Cell Res. 312, 2654–2659.[CrossRef][Medline]

Calzada, A., Hodgson, B., Kanemaki, M., Bueno, A. & Labib, K. (2005) Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev. 19, 1905–1919.[Abstract/Free Full Text]

Collins, S.R., Miller, K.M., Maas, N.L. et al. (2007) Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature. 446, 806–810.[CrossRef][Medline]

Edwards, S., Li, C.M., Levy, D.L., Brown, J., Snow, P.M. & Campbell, J.L. (2003) Saccharomyces cerevisiae DNA polymerase e and polymerase s interact physically and functionally, suggesting a role for polymerase {varepsilon} in sister chromatid cohesion. Mol. Cell. Biol. 23, 2733–2748.[Abstract/Free Full Text]

Foss, E.J. (2001) Tof1p regulates DNA damage responses during S phase in Saccharomyces cerevisiae. Genetics 157, 567–577.[Abstract/Free Full Text]

Gambus, A., Jones, R.C., Sanchez-Diaz, A., Kanemaki, M., van Deursen, F., Edmondson, R.D. & Labib, K. (2006) GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat. Cell Biol. 8, 358–366.[CrossRef][Medline]

Hanna, J.S., Kroll, E.S., Lundblad, V. & Spencer, F.A. (2001) Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion. Mol. Cell. Biol. 21, 3144–3158.[Abstract/Free Full Text]

Ho, Y., Gruhler, A., Heilbut, A. et al. (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183.[CrossRef][Medline]

Ishimi, Y. (1997) A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex. J. Biol. Chem. 272, 24508–24513.[Abstract/Free Full Text]

Kanemaki, M. & Labib, K. (2006) Distinct roles for Sld3 and GINS during establishment and progression of eukaryotic DNA replication forks. EMBO J. 25, 1753–1763.[CrossRef][Medline]

Kanemaki, M., Sanchez-Diaz, A., Gambus, A. & Labib, K. (2003) Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo. Nature 423, 720–724.[CrossRef][Medline]

Katou, Y., Kanoh, Y., Bando, M., Noguchi, H., Tanaka, H., Ashikari, T., Sugimoto, K. & Shirahige, K. (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424, 1078–1083.[CrossRef][Medline]

Labib, K., Tercero, J.A. & Diffley, J.F. (2000) Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 288, 1643–1647.[Abstract/Free Full Text]

Lei, M., Kawasaki, Y. & Tye, B.K. (1996) Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 5081–5090.[Abstract/Free Full Text]

Lengronne, A., McIntyre, J., Katou, Y., Kanoh, Y., Hopfner, K.P., Shirahige, K. & Uhlmann, F. (2006) Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell. 23, 787–799.[CrossRef][Medline]

Longtine, M.S., McKenzie, A. 3rd, Demarini, D.J., Shah, N.G., Wach, A., Brachat, A., Philippsen, P. & Pringle, J.R. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 14, 953–961.[CrossRef][Medline]

Mamnun, Y.M., Katayama, S. & Toda, T. (2006) Fission yeast Mcl1 interacts with SCFPof3 and is required for centromere formation. Biochem. Biophys. Res. Commun. 350, 125–130.[CrossRef][Medline]

Mayer, M.L., Pot, I., Chang, M., Xu, H., Aneliunas, V., Kwok, T., Newitt, R., Aebersold, R., Boone, C., Brown, G.W. & Hieter, P. (2004) Identification of protein complexes required for efficient sister chromatid cohesion. Mol. Biol. Cell 15, 1736–1745.[Abstract/Free Full Text]

Miles, J. & Formosa, T. (1992) Protein affinity chromatography with purified yeast DNA polymerase {alpha} detects proteins that bind to DNA polymerase. Proc. Natl. Acad. Sci. USA 89, 1276–1280.[Abstract/Free Full Text]

Moyer, S.E., Lewis, P.W. & Botchan, M.R. (2006) Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc. Natl. Acad. Sci. USA. 103, 10236–10241.[Abstract/Free Full Text]

Pacek, M., Tutter, A.V., Kubota, Y., Takisawa, H. & Walter, J.C. (2006) Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Mol. Cell 21, 581–587.[CrossRef][Medline]

Pan, X., Ye, P., Yuan, D.S., Wang, X., Bader, J.S. & Boeke, J.D. (2006) A DNA integrity network in the yeast Saccharomyces cerevisiae. Cell 124, 1069–1081.[CrossRef][Medline]

Petronczki, M., Chwalla, B., Siomos, M.F., Yokobayashi, S., Helmhart, W., Deutschbauer, A.M., Davis, R.W., Watanabe, Y. & Nasmyth, K. (2004) Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase-{alpha}-associated protein Ctf4 is essential for chromatid disjunction during meiosis II. J. Cell Sci. 117, 3547–3559.[Abstract/Free Full Text]

Santocanale, C. & Diffley, J.F. (1998) A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature 395, 615–618.[CrossRef][Medline]

Schlesinger, M.B. & Formosa, T. (2000) POB3 is required for both transcription and replication in the yeast Saccharomyces cerevisiae. Genetics 155, 1593–1606.[Abstract/Free Full Text]

Skibbens, R.V. (2004) Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion. Genetics 166, 33–42.[Abstract/Free Full Text]

Szyjka, S.J., Viggiani, C.J. & Aparicio, O.M. (2005) Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae. Mol. Cell 19, 691–697.[CrossRef][Medline]

Takayama, Y., Kamimura, Y., Okawa, M., Muramatsu, S., Sugino, A. & Araki, H. (2003) GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast. Genes Dev. 17, 1153–1165.[Abstract/Free Full Text]

Tan, B.C., Chien, C.T., Hirose, S. & Lee, S.C. (2006) Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication. EMBO J. 25, 3975–3985.[CrossRef][Medline]

Tourrière, H., Versini, G., Cordón-Preciado, V., Alabert, C. & Pasero, P. (2005) Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53. Mol. Cell 19, 699–706.[CrossRef][Medline]

Tsutsui, Y., Morishita, T., Natsume, T., Yamashita, K., Iwasaki, H., Yamao, F. & Shinagawa, H. (2005) Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase {alpha}: evidence for a multifunctional role of Mcl1 in DNA replication and repair. Curr. Genet. 48, 34–43.[CrossRef][Medline]

Warren, C.D., Eckley, D.M., Lee, M.S., Hanna, J.S., Hughes, A., Peyser, B., Jie, C., Irizarry, R. & Spencer, F.A. (2004) S-phase checkpoint genes safeguard high-fidelity sister chromatid cohesion. Mol. Biol. Cell 15, 1724–1735.[Abstract/Free Full Text]

Weinert T. (1998) DNA damage and checkpoint pathways: molecular anatomy and interactions with repair. Cell 94, 555–558.[CrossRef][Medline]

Williams, D.R. & McIntosh, J.R. (2002) mcl1+, the Schizosaccharomyces pombe homologue of CTF4, is important for chromosome replication, cohesion, and segregation. Eukaryot. Cell 1, 758–773.[Abstract/Free Full Text]

Williams, D.R. & McIntosh, J.R. (2005) Mcl1p is a polymerase {alpha} replication accessory factor important for S-phase DNA damage survival. Eukaryot. Cell 4, 166–177.[Abstract/Free Full Text]

Zhou, B.B. & Elledge, S.J. (2000) The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439.[CrossRef][Medline]

Zhu, W., Ukomadu, C., Jha, S., Senga, T., Dhar, S.K., Wohlschlegel, J.A., Nutt, L.K., Kornbluth, S. & Dutta, A. (2007) Mcm10 and And–1/CTF4 recruit DNA polymerase {alpha} to chromatin for initiation of DNA replication. Genes Dev. 21, 2288–2299.[Abstract/Free Full Text]

Received: 30 January 2009
Accepted: 6 April 2009




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