| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Biology, School of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
2 Graduate School of Biological Science, Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101 Japan
3 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-0814, Japan
4 Department of Ophthalmology, Nagoya University, School of Medicine, 65 Tsurumai, Naka-ku, Nagoya 466-8550, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
DNA synthesis on a primed M13 single-stranded template in the presence of purified replication proteins. However, it cannot substitute for RFC in promoting simian virus 40 DNA replication in vitro with crude fractions. These results demonstrate that the human Chl12-RFC complex is a second PCNA loader and that its roles in replication are clearly distinguishable from those of RFC. |
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
RFC is a heteropentameric protein complex composed of a large subunit (RFC1, 140 kD) and four smaller subunits (RFCs2-5, 37, 36, 40 and 38 kD, respectively). These subunits all belong to the family of AAA +ATPases, which are associated with diverse cellular activities and are distinguished by characteristic nucleotide binding motifs (Ogura & Wilkinson 2001). RFC forms an open ring structure that resembles two fingers (Shiomi et al. 2000). ATP-dependent structural changes in RFC promote the loading of the PCNA clamp onto DNA (Shiomi et al. 2000; Ellison & Stillman 2001; O’Donnell et al. 2001). Since PCNA is required for efficient synthesis by DNA polymerase , a eukaryotic replicative polymerase, both PCNA and RFC are essential for the functional assembly of the replication fork complex (Waga & Stillman 1998). Clamp and clamp loader systems are well known essential components in various DNA replication reactions (O’Donnell et al. 2001).
Recently, RFC1-like proteins such as Chl12 have been identified in other eukaryotic replication-related pathways, including Rad17 (Rad24 in budding yeast), which acts in the checkpoint signalling pathway (Zhou & Elledge 2000; O’Connell et al. 2000; Boddy & Russell 2001), and Elg1, which contributes to the maintenance of genome stability (Bellaoui et al. 2003; Ben-Aroya et al. 2003). Furthermore, these proteins have been demonstrated to form RFC-like pentameric complexes in association with RFCs2-5, referred to as Rad17-RFC, Elg1-RFC and Chl12-RFC, respectively (Green et al. 2000; Naiki et al. 2000, 2001; Kai et al. 2001; Mayer et al. 2001). Therefore, eukaryotes have at least four potential clamp loader complexes involved in reactions during and after DNA replication. In the case of Rad17-RFC, three proteins that function in the same checkpoint pathway, Rad9, Hus1 and Rad1, share amino acid sequence similarity with PCNA and form a trimeric complex termed Rad9-1-1. Electron microscopic observations have revealed that the molecular structures of Rad17-RFC and Rad9-1-1 are indistinguishable from those of RFC and PCNA (Griffith et al. 2002; Shiomi et al. 2002). Furthermore, Rad17-RFC loads Rad9-1-1 onto DNA, presumably in a 5'-recessed DNA end-dependent manner (Majka & Burgers 2003; Bermudez et al. 2003a; Ellison & Stillman 2003), indicating that Rad9-1-1 is the target clamp for the Rad17-RFC loader.
Human Chl12 was identified as a novel PCNA-binding protein by a proteomics approach (Ohta et al. 2002), and budding yeast Elg1-RFC has also been reported to physically interact with PCNA (Kanellis et al. 2003), suggesting that the two predicted loader complexes load PCNA onto DNA as well as RFC does. However, the mechanistic consequences of the interactions of PCNA with different loader complexes remain undetermined.
PCNA is the principle clamp for DNA replication but its possible involvement in sister chromosome cohesion has been suggested by its genetic interaction with a cohesion factor, Ctf7 (Skibbens et al. 1999). Since cohesion is established in S phase in parallel with the progression of the replication fork (Michaelis et al. 1997), PCNA may play a crucial role in coordinating the two processes. A novel X class DNA polymerase, DNA polymerase 
(pol ; Trf4), has been suggested to function in the cohesion pathway (Wang et al. 2000; Burgers et al. 2001) and may be responsible for DNA synthesis at cohesion sites. Thus, we hypothesize that PCNA loading mediated by Chl12-RFC has a role in converting the major replication fork complex to a pol -containing complex since there are controversial reports that pol and other X class proteins function as poly(A) polymerases rather than as DNA polymerase (Saitoh et al. 2002; Read et al. 2002).
To study the functions of Chl12-RFC and PCNA during the establishment of cohesion, we purified the human Chl12-RFC complex with a baculovirus expression system and characterized its biochemical properties. Here, we provide evidence that Chl12-RFC indeed functions as a second PCNA loader but that it can be distinguished from the replication loader RFC by its inability to promote simian virus 40 (SV40) DNA replication.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
We have previously expressed a 779 amino acid polypeptide from a human Chl12 cDNA sequence, which yielded a protein similar in size to the budding yeast Chl12 (Ohta et al. 2002). However, a recent inspection of database sequences suggested the presence of a novel start codon, 588 base pairs upstream of the previously identified open reading frame. To determine which start codon is functional in the native Chl12 gene in human cells, we compared the products of two human Chl12 cDNAs (L and S: 975 and 779 amino acid residues, respectively) with the native protein. As indicated in Fig. 1A, native Chl12 isolated from a human cell lysate by PCNA-affinity chromatography (Ohta et al. 2002) and recombinant human Chl12(L) had an apparent molecular weight of 110 kD, whereas the recombinant human Chl12(S) had a higher electrophoretic mobility. Furthermore, the FLAG-human Chl12(L) formed a pentameric complex with RFCs2-5, similar to that formed with RFC1 and Chl12(S) (Fig. 1B). Hereafter, we refer to human Chl12(L) as Chl12, and to the version that is 196 residues shorter as Chl12(S).
|
First, we studied the molecular structure of the Chl12-RFC complex in the absence of ATP by transmission electron microscopy (TEM). As shown in Fig. 2C–H, at higher magnification, the complex appeared as an oval-shaped ring with a cleft-like opening at one end. We were able to distinguish a pentameric organization in some of the better resolved pictures (Fig. 2D,E,F) reminiscent of what is observed for RFC (Fig. 2I–K) and Rad17-RFC (Shiomi et al. 2000, 2002 Griffith et al. 2002). As indicated by the tracings in Fig. 2f–h, Chl12-RFC can be superimposed on the RFC structure. The average dimensions determined from inspection of 108 images were 18.3 ± 2.1 nm and 14.8 ± 1.7 nm for the longer and shorter axes, respectively. An analysis of 76 images of RFC complexes yielded very similar values, 18.8 ± 2.0 nm and 15.4 ± 1.6 nm. The reduced dimensions for RFC compared to those based on our previous observations (Shiomi et al. 2000, 2002) are due to lighter platinum shadowing caused by a shorter evaporation time (1 min) (Shiomi et al. 2000, 2002; 1.5 min).
|
We have demonstrated that like RFC, reconstituted Chl12(S)-RFC binds to PCNA (Ohta et al. 2002), implying that Chl12-RFC may function as a PCNA loader. Since eukaryotes have multiple clamps, including PCNA and Rad9-1-1, we studied the binding specificity of these two clamps with loader complexes. FLAG-tagged RFC, Chl12-RFC and Rad17-RFC were prebound to anti-FLAG antibody affinity beads (anti-FLAG beads) and mixed with purified PCNA and Rad9-1-1, and their interactions were monitored (Fig. 3). RFC and Rad17-RFC specifically interacted with their respective target clamps PCNA and Rad9-1-1 in the presence of 0.1 M NaCl. RFC bound to PCNA 2–3 times more strongly than Rad17-RFC bound to Rad9-1-1. Binding specificity and efficiency were not affected significantly by the addition of ATP (data not shown). Chl12-RFC also specifically bound PCNA but less efficiently than did RFC, similar to the binding efficiency of Rad17-RFC to Rad9-1-1 (Fig. 3C). Chl12(S)-RFC bound to PCNA more efficiently than did Chl12-RFC, and its PCNA-binding activity was more than half that of RFC, as shown previously (Ohta et al. 2002).
|
We prepared highly purified Chl12-RFC by anti-FLAG antibody column chromatography and glycerol gradient centrifugation (Fig. 4A). Almost equimolar amounts of Chl12 and RFCs2-5 cosedimented in fractions 9 and 10 (Fig. 4B), which correspond to a molecular size of 9.0S, as was also observed for RFC and Rad17-RFC (data not shown). We detected an ATPase activity in the absence of DNA that cosedimented with Chl12-RFC and peaked in fractions 9 and 10, exactly the same profile as the protein peak. This activity was stimulated by polydA/oligodT and further enhanced by the addition of PCNA (Fig. 4C). ATP was hydrolysed in the presence of 15 ng of RFC or Chl12-RFC at a rate of 5.0 molecules/min per loader for both complexes, which increased to 18 and 10 ATP molecules/min for each respective loader when polydA/oligodT was added, and further increased to 40 and 38 ATP molecules/min when PCNA was added (Fig. 5A,B). Thus, Chl12-RFC exhibits almost the same ATPase activity in the presence of DNA and PCNA as does RFC. Chl12(S)-RFC had the same ATPase activity as Chl12-RFC (data not shown).
|
|
Chl12-RFC promotes the loading of PCNA
In further studies of Chl12-RFC activity, the ability of the complex to load PCNA onto nicked plasmid DNA was assessed using Sepharose-CL4B spin column chromatography (Iida et al. 2002). Under our assay conditions, about 10% of the input PCNA was loaded onto DNA by RFC in the presence of ATP (Fig. 6, lane 4). This activity was inhibited by the addition of ATP
S (Fig. 6, lane 5). The loading of PCNA by Chl12-RFC was similarly tested. As shown in Fig. 6, lane 7, only 0.5% of input PCNA was loaded onto DNA. The estimated amounts of PCNA loaded onto a singly nicked plasmid by RFC and Chl12-RFC were 4 and 0.2 molecules of PCNA molecules/plasmid, respectively. The Chl12-RFC activity was ATP hydrolysis-dependent (Fig. 6, lane 8), as was observed for RFC, and as reported by Bermudez et al. (2003b). These results clearly indicate that Chl12-RFC loads PCNA onto nicked circular DNA in an ATP-dependent manner, although the loading efficiency of this complex is much lower than that of RFC. A similar PCNA loading activity was detected for Chl12(S)-RFC (0.4 PCNA molecules/nicked plasmid; Fig. 6, lane 10).
|
Chl12-RFC loads a functional PCNA clamp that stimulates pol DNA synthesis but cannot substitute for RFC in the in vitro replication of SV40 DNA
The Chl12-RFC complex can physically load PCNA onto DNA. We tested whether the loaded PCNA was functional by measuring pol DNA synthesis on primed M13 ssDNA in the presence of Chl12-RFC. In the absence of a clamp or clamp-loader, pol synthesizes little DNA (Tsurimoto & Stillman 1989b). Figure 7 A shows that like RFC, Chl12-RFC supports pol DNA synthesis in a concentration-dependent manner.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
We have identified Chl12 as a novel PCNA binding protein, and demonstrated that the reconstituted Chl12(S)-RFC complex binds PCNA with almost the same efficiency as does RFC (Ohta et al. 2002). Our data indicate that Chl12, which is 196 amino acids longer than Chl12(S), has the same molecular mass as a protein in human cell lysates that is recognized by the anti-Chl12 antibody, and which corresponds to native Chl12. Chl12(S) has the same biochemical characteristics as Chl12: interaction with RFCs2-5 to form a complex that specifically binds PCNA, exhibits ATPase and DNA binding activity, and loads PCNA onto DNA. Therefore, the N-terminal 196 residues are apparently dispensable for Chl12 functions in vitro. A precise comparison showed that Chl12(S)-RFC has DNA- and PCNA-binding activities that are two- to three-fold higher than those of Chl12-RFC, correlating well with its higher PCNA loading activity. This result suggests that the N-terminal region may have a role in regulating Chl12-RFC activity. Similarly, an N-terminal truncation of RFC1 did not affect PCNA-binding and DNA-binding activities, but conferred a higher PCNA loading activity than observed for the full length RFC1 (Uhlmann et al. 1997; Podust et al. 1998), indicating the presence of a common regulatory function in the N-terminus of the large subunits of clamp loader complexes. Since the N-terminal region of RFC1 has a DNA ligase motif considered to be involved in DNA binding (Mossi & Hubscher 1998), these regions may regulate PCNA loading activity, although there are no motifs similar to the DNA ligase motif in the N-terminal of Chl12. Further analyses are necessary to identify regulatory motifs in the N-terminal regions of clamp loaders.
RFC1, Chl12 and Rad17 are variable subunits in structurally similar clamp loaders
Observations in this and previous works (Shiomi et al. 2000, 2002; Griffith et al. 2002) indicate that the pentameric complexes formed by the common subunits RFCs2-5 and the variable subunits RFC1, Rad17 or Chl12 exhibit similar molecular structures in terms of shape and size, as shown by TEM analysis. The variable subunits RFC, Rad17 and Chl12 are 140, 75, and 110 kD, respectively, with significant differences in the lengths of the N- and C-terminal regions that flank the highly conserved AAA +motif region. Since the three complexes have the same sizes, as judged by TEM, the terminal regions of the variable subunits have minimal effects on the protein structures, and the frameworks of the pentameric complexes are kept the same, even with their size differences.
In addition to overall molecular structure, the biochemical properties of these clamp loader complexes are conserved (Shiomi et al. 2002 Griffith et al. 2002; Bermudez et al. 2003a, 2003b; Merkle et al. 2003; Majka & Burgers 2003), although the target clamps for each is different, and each has a different cellular role. This finding indicates that the variable subunits of the eukaryotic clamp loaders RFC1, Rad17 and Chl12 determine their specificities for target clamps and also distinguish their own functional DNA sites and/or partner proteins to play roles as specific loader complexes.
Chl12-RFC alone can load PCNA onto DNA
Chl12-RFC loads PCNA onto nicked circular DNA in an ATP-dependent manner, but its efficiency is much lower than that of RFC in the presence of 90 mM NaCl, paralleling its low affinity for DNA under these conditions. PCNA is loaded onto RPA-coated primed M13 ssDNA with similar efficiency by Chl12-RFC and RFC, indicating that structural differences in DNA (such as nicks or gaps) and the presence or absence of RPA affect the activity of Chl12-RFC. The observation that PCNA is loaded onto primed M13 ssDNA by RFC with lesser efficiency than onto a nicked circular DNA under our assay conditions is interesting. This difference appears to be due to a limit in the number of PCNA molecules that can be loaded onto M13 ssDNA, which has a double stranded region of only 20 bp, in contrast to the almost fully double-stranded nicked circular DNA. In this respect, the inefficient loading of PCNA onto the latter substrate by Chl12-RFC may be due to its inability to load multiple PCNAs at a single DNA end. Related to this point, it has been reported that Chl12-RFC forms heptameric complexes with the essential cohesion factors Ctf8 and Dcc1 in budding yeast and human cells (Mayer et al. 2001; Merkle et al. 2003; Bermudez et al. 2003b). Bermudez et al. (2003b) have further indicated that human Chl12-RFC-Ctf8-Dcc1 has a greater PCNA loading activity than does Chl12-RFC, suggesting that Ctf8 and Dcc1 may make PCNA loading by Chl12-RFC in a more catalytic manner. It is also possible that Chl12-RFC recognizes other target DNA sites more readily than our substrate DNAs. Thus, further studies with a more native target DNA, composed of cohesion site-specific DNA structures and/or specific binding proteins (Cohen-Fix 2001; Carson & Christman 2001) are necessary to understand the mechanisms by which PCNA is loaded during the establishment of cohesion.
Chl12-RFC cannot substitute for RFC in SV40 DNA replication in vitro, although it loads functional PCNA onto DNA
Our results indicate that PCNA loaded by Chl12-RFC supports DNA synthesis by pol on a primed M13 ssDNA template at almost the same level as that supported by RFC. The question of why the two loaders are involved independently in DNA replication and chromosome cohesion pathways remains unanswered. PCNA loaded by Chl12-RFC and RFC must differ in some way, perhaps in the recruitment of specific partner(s). Indeed, in the SV40 DNA replication reaction with a crude fraction, Chl12-RFC, even in excess, did not substitute for RFC, strongly suggesting that the replication machinery can distinguish the functional clamp loader, probably through specific protein-protein interactions. DNA synthesis on a primed M13 ssDNA template with fraction I* indicates the presence of an inhibitor(s) of Chl12-RFC in the crude fraction, which may be a factor(s) that specifies RFC as the clamp loader for DNA replication. Our preliminary data suggest that discrimination occurs during the PCNA loading step by two loader proteins in the crude fraction (data not shown).
Roles of the clamp and loader system in the cohesion reaction
The involvement of PCNA in the establishment of sister chromatid cohesion is implied by its genetic interaction with the essential cohesion factor Ctf7 (Skibbens et al. 1999). A recent report indicates that Chl12-RFC also physically interacts with the cohesion establishment factors Scc1 and Smc1 in vivo (Bermudez et al. 2003b). Thus, PCNA and Chl12-RFC may be two key players in cohesion. In DNA replication, RFC and PCNA function together as a matchmaker to link DNA synthesis sites with DNA polymerases and other enzymes. Thus, Chl12-RFC and PCNA may play similar roles in cohesion reactions. The identification of target DNA structures and target partner proteins is now crucial. As described in the Introduction, one potential partner is pol , a putative cohesion-specific DNA polymerase, and the possibility that it is recruited to DNA by Chl12-RFC and PCNA is testable. Future studies with these purified components should allow elucidation of the chain of interactions essential for establishment of chromosome cohesion and its coordination with DNA replication.
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
All proteins and cDNAs described here are of human origin unless otherwise stated. Baculovirus systems expressing FLAG-RFC1, the four small RFC subunits (RFCs2-5), FLAG-Rad17, Rad9, Hus1, Rad1 and FLAG-Chl12(S), which is 196 amino acids shorter than native Chl12, were previously described (Shiomi et al. 2002; Ohta et al. 2002). Baculovirus expressing FLAG-Chl12 (derived from the full length cDNA) was constructed amplification of the N-terminal 196 amino acid-encoding region from I.M.A.G.E. clone 4156341 (Invitrogen, CA, USA) and insertion into pBacPAK-FLAG-Chl12(S).
Antibodies
The 5A10 monoclonal antibody reacting with human PCNA was purchased from MBL (Nagoya, Japan). Antisera against Chl12 and Rad1 were raised by injecting synthetic N-terminal 15 residues of each protein into rabbits (SIGMA Genosys, Hokkaido, Japan).
Pull-down assay of clamp-clamp loader interactions
Aliquots (about 0.2 µg) of FLAG-Chl12-RFC, FLAG-Chl12(S)-RFC, FLAG-RFC or FLAG-Rad17-RFC produced from Sf9 cells were prebound with 3 µl of anti-FLAG beads (anti-FLAG M2-agarose affinity beads, Sigma, USA) at 0 °C for 1 h. After 4 washes with 50 µl buffer H [25 mM HEPES (pH 7.5), 1 mM EDTA (ethylenediaminetetraacetic acid), 0.01% NP-40, 10% glycerol, 0.1 mM PMSF (phenylmethylsulphonyl fluoride), 2 µg/ml leupeptin] containing 1 M NaCl, the beads were mixed with 5, 10 or 20 ng of purified PCNA or Rad9-1-1 in 10 µl buffer H (pH 7.5) with 0.1 M NaCl at 0 °C for 10 min, washed 4 times with 10 µl of the same buffer, and treated with 20 µl 0.1 M glycine (pH 3.5) to elute the bound proteins. 10 µl each of the eluted fractions was separated in a 12.5% SDS-PAGE gel and analysed by immuno-blotting with anti-PCNA or anti-Rad1 antibodies. Co-precipitated PCNA or Rad1 was quantified using Kodak 1D scientific imaging software (Eastman Kodak Campany, CT, USA).
Preparation of FLAG-Chl12-RFC, FLAG-RFC, PCNA and Rad9-1-1 complexes
PCNA and Rad9-1-1 complexes were purified as previously published (Shiomi et al. 2000, 2002). FLAG-RFC and FLAG-Chl12-RFC were prepared from Sf9 cells (3 x 108 cells) coinfected with combinations of viruses for expression of FLAG-RFC1 and RFCs2-5 or FLAG-Chl12 and RFCs2-5, respectively. FLAG-tagged loader proteins were purified essentially as previously described for FLAG-Rad17-RFC (Shiomi et al. 2002).
Electron microscopicy
Highly purified protein samples were mixed with glycerol to a final concentration of 50%. Replica preparation and TEM observations following platinum shadowing were performed as previously described (Shiomi et al. 2000).
ATPase and DNA binding assays
ATPase and DNA binding assays were conducted according to Tsurimoto & Stillman (1990). For ATPase, 25 µM of polydA/oligodT (nucleotide molar ratio 5 : 1, Amersham Biosciences) and 300 ng of purified PCNA were added to the reaction mixture where indicated. After incubation at 37 °C for appropriate times, 32Pi-release was measured as the Norit A-unadsorbed count. For the DNA binding assay, the indicated amounts of loader proteins and 32P-labelled polydA or polydA/oligodT (nucleotide molar ratio 5 : 1) were incubated at room temperature for 15 min without nucleotides and filtered through an alkali-washed nitrocellulose membrane (Immobilon-NC, Millipore, MA, USA). The membrane-trapped radioactivity was measured as protein-bound DNA with a Fuji Image analyser BAS2000 (Fuji films, Tokyo, Japan).
Clamp-loading assay
Clamp-loading assays with nicked circular DNA were carried out essentially as described (Iida et al. 2002). Briefly, reaction mixtures (20 µl) containing 10 mM HEPES (pH 7.5), 0.2 mM EDTA, 10 mM MgCl2, 0.05% Tween 20, 2 mM ATP, the indicated concentration of NaCl, 60 ng nicked pUC118 (one nick/molecule on average), 100 ng PCNA, and 30 ng RFC or Chl12-RFC were incubated at room temperature for 15 min. To use M13 mp18 ssDNA (Takara, Japan) for a loading assay substrate instead of nicked pUC118, 75 ng of this DNA was annealed with a 3-fold molar excess of 20mer sequencing primer (5'-GTTGTAAAACGACGGCCAGT-3') in the above reaction mixture, 750 ng of RPA was included, and the amount of RFC or Chl12-RFC was increased to 90 ng. In both cases, protein-DNA complexes were recovered in the excluded fraction following two successive fractionations with Sepharose CL4B spin columns (Amersham Biosciences, USA), and half of fraction was assayed for PCNA by immuno-blot analysis with the anti-PCNA antibody, followed by quantification with Kodak 1D scientific imaging software.
Assay of pol -mediated DNA synthesis on primed M13 ssDNA
DNA synthesis with primed M13 ssDNA was carried out with slight modifications of the previous method (Tsurimoto & Stillman 1991). A reaction mixture (5 µl) containing 30 mM HEPES (pH 7.5), 7 mM MgCl2, 40 mM NaCl, 0.5 mM DTT, 0.1 mg/ml BSA (bovine serum albumin), 25 µM each dNTP with ß-[32P]-dTTP, 20 ng primed M13 ssDNA, 12.5 ng PCNA, 200 ng RPA and 15 ng pol was incubated at 37 °C for 30min, and the incorporated radioactivity was measured.
RFC complementation assay for SV40 DNA replication in vitro
RFC complementation assays with the SV40 DNA replication substrate were performed at 37 °C for 60 min in a standard reaction mixture (5 µl) containing the indicated amounts of RFC or Chl12-RFC, optimal amounts of phospho-cellulose fraction I*, purified large T antigen, topoisomerases I and II and 6 µg/ml pSVO11 (Tsurimoto & Stillman 1989a).
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Present address: aAmersham Biosciences K.K. Sanken Bldg. 3-25-1 Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan;
bDepartment of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
* Correspondence: E-mail: ttsurscb{at}mbox.nc.kyushu-u.ac.jp |
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Ben-Aroya, S., Koren, A., Liefshitz, B., Steinlauf, R. & Kupiec, M. (2003) ELG1, a yeast gene required for genome stability, forms a complex related to replication factor C. Proc. Natl. Acad. Sci. USA
100, 9906–9911.
Bermudez, V.P., Lindsey-Boltz, L.A., Cesare, A.J., et al. (2003a) Loading of the human 9–1–1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc. Natl. Acad. Sci. USA
100, 1633–1638.
Bermudez, V.P., Maniwa, Y., Tappin, I., Ozato, K., Yokomori, K. & Hurwitz, J. (2003b) The alternative Ctf18-Dcc1-Ctf8-replication factor C complex required for sister chromatid cohesion loads proliferating cell nuclear antigen onto DNA. Proc. Natl. Acad. Sci. USA
100, 10237–10242.
Boddy, M.N. & Russell, P. (2001) DNA replication checkpoint. Curr. Biol. 11, R953–R956.[CrossRef][Medline]
Burgers, P.M.J., Koonin, E.V., Bruford, E., et al. (2001) Eukaryotic DNA polymerases: Proposal for a revised nomenclature. J. Biol. Chem.
276, 43487–43490.
Carson, D.R. & Christman, M.F. (2001) Evidence that replication fork components catalyze establishment of cohesion between sister chromatids. Proc. Natl. Acad. Sci. USA
98, 8270–8275.
Cohen-Fix, O. (2001) The making and breaking of sister chromatid cohesion. Cell 106, 137–140.[CrossRef][Medline]
Ellison, V. & Stillman, B. (2001) Opening of the clamp: an intimate view of an ATP-driven biological machine. Cell 106, 655–660.[CrossRef][Medline]
Ellison, V. & Stillman, B. (2003) Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA. Blos Biol. 1, 231–243.
Green, C.M., Erdjument-Bromage, H., Tempst, P. & Lowndes, N.F. (2000) A novel Rad24 checkpoint protein complex closely related to replication factor C. Curr. Biol. 10, 39–42.[CrossRef][Medline]
Griffith, J.D., Lindsey-Boltz, L.A. & Sancar, A. (2002) Structures of the human Rad17-RFC and checkpoint Rad9–1–1 complexes visualized by glycerol-spray/low voltage microscopy. J. Biol. Chem.
277, 15233–15236.
Iida, T., Suetake, I., Tajima, S., et al. (2002) PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimethylated DNA. Genes Cells 7, 997–1007.[Abstract]
Kai, M., Tanaka, H. & Wang, T.S. (2001) Fission yeast Rad17 associates with chromatin in response to aberrant genomic structures. Mol. Cell. Biol.
21, 3289–3301.
Kanellis, P., Agyei, R. & Durocher, D. (2003) Elg1 Forms an Alternative PCNA-Interacting RFC Complex Required to Maintain Genome Stability. Curr. Biol. 13, 1583–1595.[CrossRef][Medline]
Kouprina, N., Kroll, E., Kirillov, A., Bannikov, V., Zakharyev, V. & Larionov, V. (1994) CHL12, a gene essential for the fidelity of chromosome transmission in the yeast Saccharomyces cerevisiae. Genetics 138, 1067–1079.[Abstract]
Kouprina, N., Tsouladze, A., Koryabin, M., Hieter, P., Spence, R.F. & Larionov, V. (1993) Identification and genetic mapping of CHL genes controlling mitotic chromosome transmission in yeast. Yeast 9, 11–19.[CrossRef][Medline]
Lee, J.Y. & Orr-Weaver, T.L. (2001) The molecular basis of sister-chromatid cohesion. Annu. Rev. Cell. Dev. Biol. 17, 753–777.[CrossRef][Medline]
Majka, J. & Burgers, P.M. (2003) Yeast Rad17/Mec3/Ddc1: a sliding clamp for the DNA damage checkpoint. Proc. Natl. Acad. Sci. USA
100, 2249–2254.
Mayer, M.L., Gygi, S.P., Aebersold, R. & Hieter, P. (2001) Identification of RFC (Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae. Mol. Cell 7, 959–970.[CrossRef][Medline]
Merkle, C.J., Karnitz, L.M., Henry-Sanchez, J.T. & Chen, J. (2003) Cloning and characterization of hCTF18, hCTF8, and hDCC1. Human homologs of a Saccharomyces cerevisiae complex involved in sister chromatid cohesion establishment. J. Biol. Chem.
278, 30051–30056.
Michaelis, C., Ciosk, R. & Nasmyth, K. (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91, 35–45.[CrossRef][Medline]
Mossi, R. & Hubscher, U. (1998) Clamping down on clamps and clamp loaders—the eukaryotic replication factor C. Eur. J. Biochem. 254, 209–216.[Medline]
Naiki, T., Kondo, T., Nakada, D., Matsumoto, K. & Sugimoto, K. (2001) Chl12 (Ctf18) forms a novel replication factor C-related complex and functions redundantly with Rad24 in the DNA replication checkpoint pathway. Mol. Cell. Biol.
21, 5838–5845.
Naiki, T., Shimomura, T., Kondo, T., Matsumoto, K. & Sugimoto, K. (2000) Rfc5, in cooperation with rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae. Mol. Cell. Biol.
20, 5888–5896.
Nasmyth, K. (2001) Disseminating the genome. joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu. Rev. Genet. 35, 673–745.[CrossRef][Medline]
O'Connell, M.J., Walworth, N.C. & Carr, A.M. (2000) The G2-phase DNA-damage checkpoint. Trends Cell Biol. 10, 296–303.[CrossRef][Medline]
O'Donnell, M., Jeruzalmi, D. & Kuriyan, J. (2001) Clamp loader structure predicts the architecture of DNA polymerase III holoenzyme and RFC. Curr. Biol. 11, R935–R946.[CrossRef][Medline]
Ogura, T. & Wilkinson, A.J. (2001) AAA+ superfamily ATPases: common structure—diverse function. Genes Cells 6, 575–597.[Abstract]
Ohta, S., Shiomi, Y., Sugimoto, K., Obuse, C. & Tsurimoto, T. (2002) A proteomics approach to Identify proliferating cell nuclear antigen (PCNA)-binding proteins in human cell lysates: Identification of the human Chl12/RFCs2–5 complex as a novel PCNA-binding protein. J. Biol. Chem.
277, 40362–40367.
Podust, V.N., Tiwari, N., Stephan, S. & Fanning, E. (1998) Replication factor C disengages from proliferating cell nuclear antigen (PCNA) upon sliding clamp formation, and PCNA itself tethers DNA polymerase delta to DNA. J. Biol. Chem.
273, 31992–31999.
Read, R.L., Martinho, R.G., Wang, S.W., Carr, A.M. & Norbury, C.J. (2002) Cytoplasmic poly(A) polymerases mediate cellular responses to S phase arrest. Proc. Natl. Acad. Sci. USA
99, 12079–12084.
Saitoh, S., Chabes, A., McDonald, W.H., Thelander, L., Yates, J.R. & Russell, P. (2002) Cid13 is a cytoplasmic poly(A) polymerase that regulates ribonucleotide reductase mRNA. Cell 109, 563–573.[CrossRef][Medline]
Shiomi, Y., Shinozaki, A., Nakada, D., et al. (2002) Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9–1–1 and Rad17-RFC. Genes Cells 7, 861–868.[Abstract]
Shiomi, Y., Usukura, J., Masamura, Y., et al. (2000) ATP-dependent structural change of the eukaryotic clamp-loader protein, replication factor C. Proc. Natl. Acad. Sci. USA
97, 14127–14132.
Skibbens, R.V., Corson, L.B., Koshland, D. & Hieter, P. (1999) Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery. Genes Dev.
13, 307–319.
Tsurimoto, T. & Stillman, B. (1989a) Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro. Mol. Cell. Biol.
9, 609–619.
Tsurimoto, T. & Stillman, B. (1989b) Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta. EMBO J. 8, 3883–3889.[Medline]
Tsurimoto, T. & Stillman, B. (1990) Functions of replication factor C and proliferating-cell nuclear antigen. functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. Proc. Natl. Acad. Sci. USA
87, 1023–1027.
Tsurimoto, T. & Stillman, B. (1991) Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins. J. Biol. Chem.
266, 1950–1960.
Uhlmann, F., Cai, J., Gibbs, E., O'Donnel, I.M. & Hurwitz, J. (1997) Deletion analysis of the large subunit p140 in human replication factor C reveals regions required for complex formation and replication activities. J. Biol. Chem.
272, 10058–10064.
Waga, S. & Stillman, B. (1998) The DNA replication fork in eukaryotic cells. Annu. Rev. Biochem. 67, 721–751.[CrossRef][Medline]
Wang, Z., Castano, I.B., De Las Penas, A., Adams, C. & Christman, M.F. (2000) Pol kappa: a DNA polymerase required for sister chromatid cohesion. Science
289, 774–779.
Zhou, B.B. & Elledge, S.J. (2000) The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439.[CrossRef][Medline]
Received: 19 November 2003
Accepted: 19 January 2004
This article has been cited by other articles:
|
|
 |
|
  A. Farina, J.-H. Shin, D.-H. Kim, V. P. Bermudez, Z. Kelman, Y.-S. Seo, and J. Hurwitz Studies with the Human Cohesin Establishment Factor, ChlR1: ASSOCIATION OF ChlR1 WITH Ctf18-RFC AND Fen1 J. Biol. Chem., July 25, 2008; 283(30): 20925 - 20936. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Berkowitz, K. H. Kaestner, and T. A. Jongens Germline expression of mammalian CTF18, an evolutionarily conserved protein required for germ cell proliferation in the fly and sister chromatid cohesion in yeast Mol. Hum. Reprod., March 1, 2008; 14(3): 143 - 150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. B. Ansbach, C. Noguchi, I. W. Klansek, M. Heidlebaugh, T. M. Nakamura, and E. Noguchi RFCCtf18 and the Swi1-Swi3 Complex Function in Separate and Redundant Pathways Required for the Stabilization of Replication Forks to Facilitate Sister Chromatid Cohesion in Schizosaccharomyces pombe Mol. Biol. Cell, February 1, 2008; 19(2): 595 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shiomi, C. Masutani, F. Hanaoka, H. Kimura, and T. Tsurimoto A Second Proliferating Cell Nuclear Antigen Loader Complex, Ctf18-Replication Factor C, Stimulates DNA Polymerase {eta} Activity J. Biol. Chem., July 20, 2007; 282(29): 20906 - 20914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Xu, C. Boone, and G. W. Brown Genetic Dissection of Parallel Sister-Chromatid Cohesion Pathways Genetics, July 1, 2007; 176(3): 1417 - 1429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sabbioneda, B. K. Minesinger, M. Giannattasio, P. Plevani, M. Muzi-Falconi, and S. Jinks-Robertson The 9-1-1 Checkpoint Clamp Physically Interacts with Pol{zeta} and Is Partially Required for Spontaneous Pol{zeta}-dependent Mutagenesis in Saccharomyces cerevisiae J. Biol. Chem., November 18, 2005; 280(46): 38657 - 38665. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kim, K. Robertson, K. J. L. Mylonas, F. C. Gray, I. Charapitsa, and S. A. MacNeill Contrasting effects of Elg1-RFC and Ctf18-RFC inactivation in the absence of fully functional RFC in fission yeast Nucleic Acids Res., July 21, 2005; 33(13): 4078 - 4089. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. O. Bylund and P. M. J. Burgers Replication Protein A-Directed Unloading of PCNA by the Ctf18 Cohesion Establishment Complex Mol. Cell. Biol., July 1, 2005; 25(13): 5445 - 5455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tsurimoto, A. Shinozaki, M. Yano, M. Seki, and T. Enomoto Human Werner helicase interacting protein 1 (WRNIP1) functions as a novel modulator for DNA polymerase {delta} Genes Cells, January 1, 2005; 10(1): 13 - 22. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
) or Rad9-1-1 (•) bound to the beads was determined by immunoblot analysis.
) and sum of RFCs2-5 bands (•) for each fraction. The numbers above panel A and below panel B represent fractions and ‘In’ designates the pool of anti-FLAG column fractions. Arrows and numbers at the bottom of panel A indicate the positions and S-values of the marker proteins catalase, alcohol dehydrogenase, bovine serum albumin and ovalbumin (11.3, 7.4, 4.3 and 3.7S, respectively). The positions to which marker proteins migrated and their molecular masses (kD) are indicated on the left. (C) ATPase activity measured with 0.5 µl of each glycerol gradient fraction (fraction 10 contains about 15 ng of Chl12-RFC) without added DNA (
), or in the presence of polydA/oligodT and PCNA (•). (D) DNA binding measured with 0.5 µl of each glycerol gradient fraction and 4 fmol of polydA (pda, 
), 60 (
) and 90 mM NaCl (
