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J. Biol. Chem., Vol. 277, Issue 43, 40362-40367, October 25, 2002

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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^*

Satoshi Ohta, Yasushi Shiomi, Katsunori Sugimoto^§, Chikashi Obuse, and Toshiki Tsurimoto^¶

From the  Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101, Japan and the ^§ Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-0814, Japan

Received for publication, June 21, 2002, and in revised form, August 7, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Proliferating cell nuclear antigen (PCNA), a eukaryotic DNA replication factor, functions not only as a processivity factor for DNA polymerase  but also as a binding partner for multiple other factors. To understand its broad significance, we have carried out systematic studies of PCNA-binding proteins by a combination of affinity chromatography and mass spectrometric analyses. We detected more than 20 specific protein bands of various intensities in fractions bound to PCNA-fixed resin from human cell lysates and determined their peptide sequences by liquid chromatography and tandem mass spectrometry. A search with human protein data bases identified 12 reported PCNA-binding proteins from both cytoplasmic (S100 lysate) and nuclear extracts with substantial significance and four more solely from the nuclear preparation. CHL12, a factor involved in checkpoint response and chromosome cohesion, was a novel example found in both lysates. Further studies with recombinant proteins demonstrated that CHL12 and small subunits of replication factor C form a complex that interacts with PCNA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Proliferating cell nuclear antigen (PCNA)¹ was first identified as a DNA elongation factor in simian virus 40 DNA replication in vitro (1). It has a characteristic ring structure that can encircle double-stranded DNA (2, 3). Replication factor C (RFC) functions to load PCNA by opening the ring temporarily through its ATPase activity (4-6). On loading, PCNA associates with DNA polymerase (pol) , which stabilizes the interaction with template DNA and slides along it stably. Thus, it is called a "DNA sliding clamp" and functions as the processivity factor for pol  (7).

Recent studies have indicated that, in addition to pol , PCNA specifically interacts with various factors involved in cell cycle control, DNA repair, DNA methylation, and chromatin assembly, in some cases stimulating their activities (8-10). Thus, in addition to the role as the processivity factor for pol , PCNA on replicating DNA may function to recruit other factors and promote specific interactions. This suggests that a moving replication fork complex may actively accumulate factors necessary for maintenance or reorganization of replicating chromosomal structures, with PCNA playing a central role. One characteristic feature of PCNA-binding proteins is the presence of an amino acid stretch named the PCNA-interacting protein box (9) in their N- or C-terminal regions. Indeed, some of them have been identified by detection of predicted PCNA-interacting protein motifs (11, 12).

So far, more than 10 PCNA-binding proteins have been found by various means. For example, interactions of MutL homolog 1, MSH2, and myeloid cell leukemia 1 with PCNA were detected in yeast two-hybrid systems, and interactions of Gadd45, cyclin D, and p21^waf1 with PCNA were detected by co-immunoprecipitation (13-16). Most of them were, however, independently studied, and characterization of their interactions with PCNA was limited. To evaluate their importance for cellular functions, it is necessary to demonstrate their PCNA binding nature on a common background. Affinity chromatography with a PCNA fixed resin has proved a useful tool for isolating PCNA-binding proteins from cell lysates (17, 18). Indeed, one missing subunit of DNA polymerase  in mammals was identified by this method (18). However, use of classical amino acid sequencing or immunoblotting with antibodies against predictable factors may detect only a limited number of components. Thus, it is crucial to apply a broader approach, which is nevertheless still highly sensitive.

Recent advances in mass spectrometry now allow identification of proteins in limited amounts of material very efficiently (19) and make it possible to detect all proteins included in one slot of an electrophoresis gel without selection. We have applied liquid chromatography and tandem mass spectrometry (LC/MS/MS) analyses to identify components recovered by PCNA affinity chromatography. It should be noted that because we have already accumulated significant data for PCNA-binding proteins by various methods, the reliability of our approach using mass spectrometry can be easily evaluated. It is clearly useful to build up a systematic strategy to search for proteins associated with targets in cell lysates, and we have determined peptide sequences from 50 slices of SDS-polyacrylamide gels after separation of proteins in PCNA-bound fractions. A search with protein data bases revealed nearly 20 PCNA-binding proteins, including CHL12 as a novel example, identified with high reliability scores.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Preparation of Proteins-- Cytoplasmic (S100 lysate) and nuclear extracts from human 293 cells were prepared as described earlier (20). Purification steps for human and budding yeast PCNAs were detailed previously (21).

Human CHL12 (CHL12) cDNA was amplified from a gt10 HeLa cell cDNA library (CLONTECH) by the polymerase chain reaction using Pyrobest DNA polymerase (Takara) and inserted into the BamHI-EcoRI sites of pBacPAK8 to obtain pBacPAK8-CHL12. A FLAG epitope cassette was prepared by annealing two synthesized oligonucleotides (5'-GATCCATGGACTACAAGGATGACGATGACAAGA-3' and 5'-GATCTCTTGTCATCGTCATCCTTGTAGTCCATG-3'; SAWADY Technology) and inserted into the BamHI site flanking the start codon of CHL12 to yield pBacPAK8-Flag-CHL12, arranged to express the FLAG epitope-tagged CHL12 (FLAG-CHL12). These plasmid DNAs were transfected into Sf9 cells to prepare baculoviruses to express gene products. Baculoviruses for human RFC subunits (RFC1-5) and preparation of the insect cell lysates were described in Ref. 22. Preparation of the FLAG epitope-tagged RFCp140 (FLAG-p140) will be described elsewhere.

PCNA- and BSA-fixed Resin Affinity Chromatographies-- We prepared PCNA and BSA columns following a published method (23). Twenty mg of purified human PCNA or 40 mg of BSA (Takara) in 25 mM MES, pH 6.0, 100 mM NaCl, 0.01% Nonidet P-40 were cross-linked to 5 ml of Affi-Gel 15 (Bio-Rad) at 4 °C for 6 h. After removing the supernatant, the beads were incubated with 20 ml of 0.1 M ethanolamine-HCl, pH 8.2, at 4 °C overnight and washed several times with 50 mM Tris-HCl, pH 7.5. Tandemly joined BSA (1 ml) and PCNA (2 ml) columns were equilibrated with buffer A (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.01% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin) containing 0.1 M NaCl. Cytoplasmic or nuclear extracts obtained from 2 × 10¹⁰ human 293 cells were loaded onto the columns and washed with 40 ml of buffer A containing 0.1 M NaCl. Bound proteins were eluted from the PCNA column with 20 ml of buffer A containing 0.3 M NaCl. We performed two independent analyses with 293 cell S100 lysates and obtained essentially the same results.

SDS-PAGE-- The proteins were mixed with equal volumes of 2× Laemmli SDS sample buffer (24), separated in 12.5% or 4-20% SDS-polyacrylamide gels (Tefco) with running buffer (25 mM Tris, 20 mM glycine, 0.1% SDS), and stained with Coomassie Brilliant Blue (Nacalai Tesque).

Mass Spectrometry-- Proteins in the PCNA-bound fraction were applied to wells (4-mm width) of 12.5% SDS-polyacrylamide gels (1-mm thickness and 6-cm length) and separated with a 40 mA constant current for 60 min. The area from the top to the bottom of the separation gel corresponding to a molecular mass from about 300 to 30 kDa was cut essentially at 1-mm intervals (some slices were wider because of the absence of any prominent bands at those positions). The proteins in each gel slice were subjected to reduction with 10 mM dithiothreitol, alkylation with 55 mM iodoacetanide, and tryptic digestion with 12.5 µg/ml modified trypsin (Roche Molecular Biochemicals) at 37 °C for 14 h. After in gel digestion, the product peptides were extracted with 5% formic acid and acetonitrile, dried under a vacuum, and dissolved in 5% formic acid. Multiple digested peptides were obtained from a single run of each gel slice sample by microcapillary C18 reverse phase chromatography (200 µm × 5 cm capillary; Michrom BioResources, Inc.) and directly applied into an LCQ Deca quadrapole ion trap mass spectrometer (Finnigan) with a nanoelectrospray needle (New Objective) mounted on a three-dimensional stage (AMR, Tokyo, Japan). The ion trap was programmed to carry out two successive scans consisting of the first full scan MS over the range 300-2000 m/z and the second data-dependent scan of the most abundant ion in the first scan. Automatic MS/MS spectra were obtained from the highest peak in each full scan by setting a relative collision energy of 30% and an exclusion time of 5 min for molecules of the same m/z value range.

Data Base Search and Data Processing-- We searched the NCBI nonredundant protein data base with the Mascot program (Matrix Science) for high fits with the ion spectrum data generated by LC/MS/MS. The obtained raw results demonstrated the top 20 candidates for each spectrum. We examined their automatic ordering manually in terms of their reliability scores and MS spectrum profiles to pick up only highly reliable peptide data (sorted data). Included were membrane proteins, metabolic enzymes, ribosomal proteins, splicing factors, heat shock proteins, and some filament proteins, which remained in the column even after extensive washes because of their abundance or low solubility. They were deleted from the list prior to the analyses as contaminating proteins in this experimental system. The proteins in Tables I and II, designated "primary data" are the remaining examples.

Pull-down Assay with FALG-CHL12-- Sf9 cells were infected with baculoviruses arranged to express FLAG-CHL12 alone, FLAG-CHL12 and the four small subunits of RFC (RFCs2-5), or FLAG-p140 and RFCs2-5. 50-µl aliquots of cell lysates containing 0.5 mg of proteins were incubated with 3 µl of anti-FLAG M2-agarose affinity gel (Sigma) at 0 °C for 1h. After four washes with 50 µl of buffer H (25 mM HEPES-NaOH, pH 7.5, 1 mM EDTA, 10% glycerol, 0.01% Nonidet P-40, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin) containing 1 M NaCl, beads bound to about 500 ng of FLAG-CHL12, FLAG-CHL12/RFCs2-5 or FLAG-p140/RFCs2-5 complexes were obtained, further incubated with purified human or yeast PCNAs in 10 µl of buffer H containing 0.1 M NaCl at 0 °C for 1 h, and washed four times with the same buffer. The bound proteins were eluted in 20 µl of 10 mM glycine (pH 3.5). One-third of the eluates was analyzed by silver staining (Dai-ichi Pure Chemicals) following separation on a 4-20% gradient SDS-PAGE gel (Tefco).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Fractionation of Human Cell Lysates by PCNA-fixed Resin Affinity Chromatography-- More than 10 PCNA-binding proteins have been reported, with various histories of identification. Thus, some systematic approach to study PCNA-binding proteins on the same background, for example by a biochemical method, is necessary. Previous affinity chromatography attempts with native PCNA-fixed matrixes efficiently and specifically isolated several PCNA-binding proteins (17, 18), but efforts to identify the components were limited. We have therefore used the same approach in combination with highly sensitive LC/MS/MS analyses to identify PCNA-binding proteins from human cell lysates. Because the previous studies used only cytoplasmic extract from calf thymus (17) and a lysate prefractionated by ion exchange chromatography (18), some bias was generated in the primary samples. To avoid this problem, we applied S100 lysates and nuclear extracts from human 293 cells separately for systematic analyses, expecting in this way to obtain information on the distribution of PCNA-binding proteins. It should be noted that differential fractionation of proteins in the S100 lysate (referred to as the cytoplasmic extract) and nuclear extracts from 293 cells does not necessarily imply cellular localization in cytoplasm and nuclei, because significant fractions of replication proteins in nuclei are known to leak out into S100 lysates under the conditions used (20). Thus, the S100 lysate may contain nuclear proteins associating weakly with nuclear structures, in addition to authentic cytoplasmic proteins, whereas those remaining in the nuclear extract can be considered to associate more tightly with nuclear structures.

When we loaded the passed through sample from a BSA column onto a PCNA column at 0.1 M NaCl and eluted the bound components with 0.3 M NaCl buffer, more than 20 bands were specifically detected in the Coomassie Brilliant Blue-stained 12.5% SDS-polyacrylamide gels (Fig. 1). The protein bands were specific for PCNA because they were hardly detected in BSA column eluates (data not shown) and were more numerous in the nuclear than S100 lysate. Taking their molecular masses and relative intensities into consideration, strong bands corresponding to DNA polymerase  subunits, RFC subunits and Fen1 were evident where predicted.

View larger version (75K): [in this window] [in a new window]   Fig. 1.   Resolution of PCNA-bound proteins on Coomassie Brilliant Blue-stained SDS-PAGE. S100 lysate (S100) or nuclear extract (NE) obtained from human 293 cells were loaded onto PCNA columns as described under "Experimental Procedures." The eluates from 18 mg of S100 lysate corresponding to 4 × 108 cells or from 5.4 mg of nuclear extract corresponding to 2 × 108 cells were separated in 12.5% SDS-PAGE gels, and the proteins were visualized by CBB. Molecular mass markers (New England Biolabs) were run on both sides as protein size and amount standards (indicated by their molecular masses). The 66-kDa bovine serum albumin band corresponds to 0.5 µg of protein. The ladders and numbers indicate gel slices applied for mass spectrometry and correspond with the gel slice numbers in Table I.

LC/MS/MS Analyses and Search for PCNA-binding Proteins from Data Bases-- Because our purpose was to identify PCNA-binding proteins without any bias, we focused on all components recovered in the fraction. The gel area corresponding to molecular masses from 300 to 30 kDa was analyzed with LC/MS/MS as described under "Experimental Procedures." The obtained raw data were further processed to determine highly scored fits by a NCBI nonredundant protein data base search. The resulting sorted data were further organized by removing various contaminating proteins. We designated the resulting list as "primary data" and evaluated components in terms of their possible interaction with PCNA.

From the S100 lysate, we picked up 41 proteins with redundancy, 19 being independent (Table I). In the list were included 12 known PCNA-binding proteins (Table III, group 1). Among the remaining seven, only CHL12 has a known functional relation with PCNA, and we could obtain additional data in support of an actual interaction as described below. Thus, we included CHL12 in group 2 of Table III, listing potential PCNA-binding proteins or proteins with some functional relevance.

From analyses of the nuclear extract, we obtained 52 proteins in the primary data, 31 being independent members (Table II). Among them, 16 known PCNA-binding proteins were identified as members of group 1 in Table III. Eight other proteins, including CHL12, were picked up from the primary data with a relation to DNA repair or potential interaction with PCNA (group 2) in line with earlier reports (17, 25, 40). Consequently, PCNA-binding proteins seem to be enriched in the nuclear extract.

Six and seven proteins in the S100 lysate and nuclear extract, respectively, remain to be characterized and were excluded from the list of PCNA-binding proteins in Table III. Most of their functions are unknown, according to their annotations in the data base, and also there has been no literature, to our knowledge, indicating any relation with PCNA. Further studies on their amino acid sequences did not reveal any PCNA binding motifs, and we consider them to have been retained on the column independently of interaction with PCNA.

Behavior of PCNA-binding Proteins on PCNA Affinity Chromatography-- In total, 16 known PCNA-binding proteins were picked up in our experiment, and some of them have been demonstrated to be PCNA-binding proteins by similar affinity chromatography. However, DNA cytosine 5-methyltransferase, MSH3, and uracil-DNA glycosylase 2, found to interact with PCNA by other methods (26-28), were newly recovered from the affinity resin. Because further studies on their interactions were limited after the original reports, our data for specific interaction with PCNA provide strong support for the initial observations.

In addition, we could obtain several insights into the behavior of members of the list. MSH2 and some small subunits of RFC are likely to be recovered by indirect association with PCNA, because they are subunit components of their respective functional complexes and known not to have any obvious PCNA binding activity by themselves. MSH2 associates with MSH6 or MSH3 and forms distinct complexes of human MutS  and , respectively (29). Because MSH3 was only detected in the nuclear extract, we can suggest that MutS  and  are associated with different structures in human cell nuclei in line with their different substrate DNA specificities (30). Three more factors, DNA polymerase , DNA cytosine 5-methyltransferase, and RFCp140, were only identified in the fractions from the nuclear extract, suggesting particularly tight association with chromatin or some nuclear matrix elements.

Identification of CHL12 as a Novel PCNA-binding Protein-- Among the searched proteins, CHL12 is the only example not previously reported as a PCNA-binding protein, which could be judged as a novel candidate. The reasons for this judgment are as follows. First, CHL12 could be isolated from both extracts as with other authentic PCNA-binding proteins. Second, it has been suggested that CHL12 functions as a novel clamp loader protein by association with four small subunits of RFC (31, 32). A similar potential clamp loader composed of four small subunits of RFC and Rad17 has been reported and suggested to have a role in a checkpoint response pathway (33). In the case of CHL12, this potential novel clamp loader might be involved in the checkpoint response and sister chromatid cohesion pathways (31, 32). Thus, it is reasonable that it would bind to PCNA, if this is the target clamp. A study of budding yeast genetics demonstrated that PCNA actually has a role in its cohesion pathway (34).

To confirm our predictions, we assessed whether CHL12 forms a complex with RFCs2-5 and, if so, whether the complex interacts with PCNA (Fig. 2). FLAG-tagged human CHL12 was expressed in Sf9 cells and recovered with anti-FLAG antibody beads (lane 5). Upon co-expression of RFCs2-5 with FLAG-CHL12, a pentameric complex was formed, as with authentic RFC subunits (lanes 2 and 6). To test PCNA binding activity, anti-FLAG antibody beads bound to RFC or CHL12/RFCs2-5 complexes were further incubated with purified human PCNA. We could precipitate PCNA with CHL12/RFCs2-5 at almost the same level as with RFC, as shown in lanes 4 and 6. This binding is species-specific and PCNA clamp-specific, because budding yeast PCNA or another potential clamp complex, Rad9-Rad1-Hus1, hardly interacted with CHL12/RFCs2-5 (lane 9 and data not shown). It is also specific for the CHL12/RFCs2-5 complex, because beads bearing only CHL12 did not retain any human PCNA (lane 8). This result strongly supports the idea that CHL12/RFCs2-5 functions as a novel clamp loader protein. It also suggests that PCNA could be one of its target clamps. This point must be tested in future studies.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Interaction of the CHL12/RFCs2-5 complex and PCNA. The proteins precipitated with anti-FLAG antibody beads were separated on a 4-20% gradient SDS-PAGE gel and stained with silver. The samples incubated with the beads are lysates from cells expressing FLAG-CHL12 and RFC2-5 (lanes 6, 9, and 11). Purified human PCNA (250 ng) was further added to the samples in lanes 4 and 9, and 500 ng of budding yeast PCNA was added to the sample in lane 11. Purified RFC (90 ng), human PCNA (hPCNA, 25 ng), and budding yeast PCNA (scPCNA, 50 ng) were run in lanes 1, 3, 7, and 10, respectively, as standards. We confirmed four bands that appeared from 36 to 40 kDa in FLAG-CHL20 precipitates to be RFC p37, p36, p40, and p38, respectively by mass spectrometric analyses (data not shown). Recovered proteins are indicated on the right, and the molecular mass markers (in kDa) are on the left. Two bands at 69 and 59 kDa, marked with asterisks on the right of both panels, were proteins precipitated nonspecifically from cell lysates. Because we used more lysates for CHL12 (right panel) than for RFC (left panel) because of their different expression levels in insect cells, the contaminated bands appeared more strongly in the former case.

RFCp140, the largest RFC subunit, was identified only in the fraction from a nuclear extract, consistent with the previous report that functional RFC is much abundant in human nuclear extracts (35). Interestingly, the other four small subunits were also present in the S100 lysate. One reason for the different distribution of RFC subunits might be that a complex composed of only the four small subunits of RFC is present in the S100 lysate, and this can interact with PCNA. Actually, a subcomplex of RFC composed of RFC p40, p37, and p36 has been shown to form and have some affinity with PCNA (36). However, we propose an alternative explanation that these four subunits are in a complex with CHL12 and interact with PCNA as demonstrated in Fig. 2. CHL12 exhibited high scores and numbers of recovered peptides in a Mascot program search (Table I), indicating a relative abundance in the PCNA-bound fractions. Thus, the amounts of the recovered CHL12 and RFC subunits are in line with interpretation as a complex. However, our data do not rule out the possibility that RFCs2-5 exists in both conditions in the S100 lysates.

Factors Potentially Involved in the PCNA Network-- We listed seven more proteins in group 2 of Table III from the nuclear extract because of their possible relations with PCNA. Among them, Myb-binding protein 1a was recently added to the group based upon the discovery of its amino acid sequence similarity with budding yeast DNA polymerase  (Sc pol ), the fifth essential DNA polymerase involved in rRNA synthesis (40). Sc pol  has a putative PCNA-binding motif from amino acids 328-343 and is stimulated by PCNA. Thus, the presence of Myb-binding protein 1a in a PCNA-bound fraction strongly suggests functional interaction between the pol -related protein and PCNA in human cells. However, because human Myb-binding protein 1a does not conserve any DNA polymerase domains found in Sc pol , further studies will be necessary to confirm its interaction with human PCNA.

None of the remaining six proteins have any obvious PCNA-binding motifs and have not demonstrated direct interaction with purified PCNA. Thus, they may be components of functional complexes. Two of them, nuclear DNA helicase II and RPA1 had earlier been identified by immunoblotting after similar chromatography (17). Nuclear DNA helicase II has both DNA and RNA helicase activity, but no data indicating involvement in either DNA replication or transcription have yet been reported. Thus, the functional significance of its possible link with PCNA has to await future studies.

In the case of RPA, its abundance in the S100 lysates and nuclear extracts was similar, although RPA1 was recovered only from the nuclear extract. Related to this point, we picked up the RuvB-like protein 2 (RUVBL2), a TATA-binding protein-interacting protein, in the PCNA-bound fractions. Eukaryotes have two RuvB-like proteins, RUVBL1 and 2, which function as a DNA helicase in a complex (37). They are encoded by essential genes and have a role in transcription. Furthermore, RUVBL1 has been isolated as a RPA3 (the smallest subunit)-interacting protein by yeast two-hybrid assay (38). These data may suggest that RPA, RUVBL2, and some other factors, which can associate with PCNA, are recovered together in the PCNA-bound fraction as components of a transcription complex.

DNA-dependent protein kinase and Ku70/80, recovered in the PCNA-bound fractions, also form a complex and function in double-stranded DNA break repair. Ku70/80 and PCNA co-purify with RNA polymerase I complex from rat cells (25), and in addition, Ku70/80 has been reported to localize similarly with PCNA in the nucleolus (39). Thus, their identification in the PCNA-bound fractions is consistent with previous observations. In this case, involvement of PCNA in rDNA transcription is suggested.

Taken together, the evidence suggests that repair and transcription complexes include factors that interact with PCNA through binding of one or more other components. The results point to the importance of this type of proteomics approach to elucidate complicated features of protein networks, which cover the overlapping areas of DNA replication, repair, and transcription.

In conclusion, we could identify 16 PCNA-binding proteins from two types of human cell lysate by a combination of affinity chromatography with PCNA-fixed resin and highly sensitive mass spectrometric analyses. Because the identified proteins include most reported PCNA-binding proteins, the reliability and efficiency of this approach appear quite high. Therefore, proteins directly binding to a target protein can be reproducibly identified by our strategy. Furthermore, indirectly interacting components can also be efficiently isolated, as indicated for repair- and transcription-related proteins in the PCNA-bound fraction. These results strongly suggest that PCNA is involved in a large functionally linked protein network covering various cell functions in nuclei. Under the present conditions, we identified CHL12 as a novel candidate PCNA-binding protein, providing evidence that it forms a complex with RFCs2-5, which actually interacts with PCNA as strongly as RFC. Thus, PCNA potentially has two loader proteins, which may be responsible for links with multiple partners.

	ACKNOWLEDGEMENTS

We thank Dr. Waga (Osaka University) for critical reading of this manuscript and valuable comments and Drs. Takao Kawakami, Toshihide Nishimura (GlaxoSmithKline, Tsukuba laboratories in Japan), and Yasuhiro Kasahara (Naist) for valuable suggestions regarding LC/MS/MS analyses.

	Note Added in Proof

CHL12, which we expressed in this paper, was an 87-kDa (779 amino acids) protein. Its open reading frame was taken from the longest one available in a previous data base. However, the latest version of CHL12 cDNA submitted recently (Accession no. BC006278) demonstrates a longer open reading frame (975 amino acids or longer) than we used. Indeed, our mass spectrometric data (Fig. 1 and Tables I and II) indicate that CHL12 will be about a 120-kDa protein. This suggests that the native human CHL12 will have an N-terminal extension from our expressed protein. It should be noted that our 87-kDa CHL12 was sufficient to form a complex with RFCs2-5 and to interact with PCNA.

	FOOTNOTES

^* This work was supported by grants-in aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 81-743-72-5511; Fax: 81-743-72-5519; E-mail: turimoto@bs.aist-nara.ac.jp.

Published, JBC Papers in Press, August 8, 2002, DOI 10.1074/jbc.M206194200

	ABBREVIATIONS

The abbreviations used are: PCNA, proliferating cell nuclear antigen; LC/MS/MS, liquid chromatography and tandem mass spectrometry; MSH, MutS homolog; RFC, replication factor C; RPA, replication protein A; pol, polymerase; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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