Identification of a Novel Protein, PDIP38, That Interacts with the p50 Subunit of DNA Polymerase δ and Proliferating Cell Nuclear Antigen
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m208694200
ISSN1083-351X
AutoresLi Liu, Esther Rodríguez‐Belmonte, Nayef A. Mazloum, Bin Xie, Marietta Lee,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase δ (pol δ). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol δ heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol δ was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol δ and with PCNA was confirmed by pull-down assays using glutathioneS-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol δ was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol δ isolated by immunoaffinity chromatography. The association of PDIP38 with pol δ could also be demonstrated by native gel electrophoresis. The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase δ (pol δ). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol δ heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol δ was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol δ and with PCNA was confirmed by pull-down assays using glutathioneS-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol δ was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol δ isolated by immunoaffinity chromatography. The association of PDIP38 with pol δ could also be demonstrated by native gel electrophoresis. DNA polymerase polymerase δ-interacting protein proliferating cell nuclear antigen expressed sequence tag data base non-redundant nucleotide data base phenylmethylsulfonyl fluoride amino acid glutathione S-transferase open reading frame DNA replication is essential not only for duplication of the genome but also for maintenance of genomic integrity during DNA repair (1Hubscher U. Giovanni M. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (584) Google Scholar, 2Sutton M.D. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8342-8349Crossref PubMed Scopus (153) Google Scholar). Chromosomal DNA replication in eukaryotic cells requires three distinct DNA polymerases-α, -δ, and -ε (1Hubscher U. Giovanni M. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (584) Google Scholar, 2Sutton M.D. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8342-8349Crossref PubMed Scopus (153) Google Scholar, 3Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1388) Google Scholar, 4Lee M.Y.W.T. Tan C.-K. Downey K.M. So A.G. Biochemistry. 1984; 23: 1906-1913Crossref PubMed Scopus (174) Google Scholar, 5MacNeill S.A. Moreno S. Reynolds N. Nurse P. Fantes P.A. EMBO J. 1996; 15: 4613-4628Crossref PubMed Scopus (85) Google Scholar, 6Zlotkin T. Kaufmann G. Jiang Y. Lee M.Y.W.T. Uitto L. Syvaojo J. Dornreiter I. Fanning E. Nethanel T. EMBO J. 1996; 15: 2293-2305Crossref Scopus (111) Google Scholar). pol1 δ is required for replication of the leading strand and for completion of the lagging strand synthesis at the replication fork (7Waga S. Stillman B. Nature. 1994; 369: 207-212Crossref PubMed Scopus (493) Google Scholar). The action of pol δ as a processive enzyme requires its interaction with proliferating cell nuclear antigen (PCNA), which functions as a molecular sliding clamp (1Hubscher U. Giovanni M. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (584) Google Scholar). The core mammalian pol δ enzyme consists of a tightly associated heterodimer of 125- and 50-kDa subunits. pol δ has been shown recently (8Zuo S. Gibbs E. Kelven Z. Wang T.S.–F. O'Donnell M. MacNeill S.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11244-11249Crossref PubMed Scopus (70) Google Scholar, 9Reynolds N. Watt A. Fantes P.A. MacNeill S.A. Curr. Genet. 1998; 34: 250-258Crossref PubMed Scopus (50) Google Scholar, 10Gerik K.J. Li X. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 11Zhang P. Mo J. Perez A. Leon A. Liu L. Mazloum N. Xu H. Lee M.Y.W.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Hughes P. Tratner I. Ducoux M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar, 13Mo J. Liu L. Rodriquez-Belmonte E.M. Lee M.Y.W.T. Biochemistry. 2000; 39: 7245-7254Crossref PubMed Scopus (49) Google Scholar, 14Liu L. Mo J. Rodriquez-Belmonte E.M. Lee Marietta Y.W.T. J. Biol. Chem. 2000; 275: 18739-18744Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) to consist of at least four subunits, consisting of the core enzyme and two additional subunits in both yeast and mammalian systems. In the yeast Schizosaccharomyces pombe, Cdc27 and Cdm1 have been identified as the third and the fourth pol δ subunits, respectively, (8Zuo S. Gibbs E. Kelven Z. Wang T.S.–F. O'Donnell M. MacNeill S.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11244-11249Crossref PubMed Scopus (70) Google Scholar, 9Reynolds N. Watt A. Fantes P.A. MacNeill S.A. Curr. Genet. 1998; 34: 250-258Crossref PubMed Scopus (50) Google Scholar). In Saccharomyces cerevisiae Pol32p has been identified as the homologue of the S. pombe third subunit (10Gerik K.J. Li X. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). A human homologue of Cdc27, the KIAA0039 gene product (11Zhang P. Mo J. Perez A. Leon A. Liu L. Mazloum N. Xu H. Lee M.Y.W.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Hughes P. Tratner I. Ducoux M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar, 13Mo J. Liu L. Rodriquez-Belmonte E.M. Lee M.Y.W.T. Biochemistry. 2000; 39: 7245-7254Crossref PubMed Scopus (49) Google Scholar), and p12, a human homologue of Cdm1 (14Liu L. Mo J. Rodriquez-Belmonte E.M. Lee Marietta Y.W.T. J. Biol. Chem. 2000; 275: 18739-18744Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), have recently been identified and can be considered to be the third and fourth subunits of human pol δ. Our laboratory has been interested in the identification of additional protein components that are involved in the formation of the pol δ replication complex. In this study we report the identification of two novel proteins, PDIP38 and PDIP46, that interact with the p50 subunit of pol δ by the use of the yeast two-hybrid (15Fields S. Song O. Nature. 1989; 340: 245-247Crossref PubMed Scopus (4822) Google Scholar,16Hollenberg S.M. Sternganz R. Chang P.-F. Weintraub H. Mol. Cell. Biol. 1995; 5: 3813-3822Crossref Scopus (582) Google Scholar) screening method. PDIP38 was shown to be a PCNA-binding protein, and its interaction with pol δ was established by additional experiments.DISCUSSIONThe motivation for these studies was to identify novel proteins that interact with pol δ using the yeast two-hybrid system. pol δ, rigorously isolated from mammalian tissues, has been shown to consist of a tightly associated heterodimer of the p125 and p50 subunits. Two less tightly associated subunits, p68 and p12, have recently been identified as homologues of the so-called third and fourth subunits inS. pombe (11Zhang P. Mo J. Perez A. Leon A. Liu L. Mazloum N. Xu H. Lee M.Y.W.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Hughes P. Tratner I. Ducoux M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar, 13Mo J. Liu L. Rodriquez-Belmonte E.M. Lee M.Y.W.T. Biochemistry. 2000; 39: 7245-7254Crossref PubMed Scopus (49) Google Scholar, 14Liu L. Mo J. Rodriquez-Belmonte E.M. Lee Marietta Y.W.T. J. Biol. Chem. 2000; 275: 18739-18744Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In S. cerevisiae, evidence for the presence of the third subunit has been obtained, but so far a homologue of the S. pombe fourth subunit (10Gerik K.J. Li X. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) has not been identified. A yeast two-hybrid screen of a human cDNA library using the p125 and p50 subunits was carried out. Our yeast two-hybrid screening using p50 as the bait revealed three positives. Two of these were proteins of unknown function, and the third was identified as p21.Obviously, protein-protein interactions revealed by the yeast two-hybrid system do not necessarily indicate that the native (non-fusion) proteins are capable of interaction, nor do they provide any evidence that such interactions take place in a cellular context. In these studies, we have further characterized the interaction of PDIP38 with the p50 subunit of pol δ.The ability of PDIP38 to interact with p50 and also with PCNA was demonstrated by coimmunoprecipitation and GST pull-down assays. Pull-down experiments using bacterially expressed p50 and PCNA established that the interactions were direct. These experiments were important for the validation of the coimmunoprecipitation experiments from cell extracts. The coimmunoprecipitation experiments revealed that PDIP38 interacts with the pol δ complex in cell extracts, in that we could show the coimmunoprecipitation of p125 as well as p50. The results indicate that PDIP38 is able to bind to p50 when the latter is in association with p125. More significantly, an association of PDIP38 with pol δ activity during immunoaffinity chromatography was established. An association of PDIP38 with pol δ was also demonstrated by native gel electrophoresis of 293 cell lysates. These studies strongly support the view that the interaction of PDIP38 with the pol δ complex is physiological, i.e. that this interaction takes place in a cellular context and is likely to play some physiological relevant function. The issues of whether it is an accessory protein for pol δ or could be considered to be a subunit of pol δ is currently under investigation.Whereas the functions of PDIP38 are still unknown, our studies have shown that it possesses one property that is of relevance to the functions of pol δ, viz. the ability to bind to PCNA. PCNA has been shown to bind a number of proteins through a short peptide motif (18Gibbs E. Kelman Z. Gulbis J.M. O'Donnell M. Kuriyan J. Burgers P.M.J. Hurwitz J. J. Biol. Chem. 1997; 272: 2373-2381Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 19Warbrick E. Lane D.P. Glover D.M. Cox L.S. Oncogene. 1997; 14: 2313-2321Crossref PubMed Scopus (134) Google Scholar, 20Cox S.L. Trends Cell Biol. 1997; 7: 493-498Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 21Warbrick E. Heatherington W. Lane D.P. Glover D.M. Nucleic Acids Res. 1998; 26: 3925-3933Crossref PubMed Scopus (74) Google Scholar), exemplified by that present in p21Waf1 which binds to the interdomain connector loop of PCNA (18Gibbs E. Kelman Z. Gulbis J.M. O'Donnell M. Kuriyan J. Burgers P.M.J. Hurwitz J. J. Biol. Chem. 1997; 272: 2373-2381Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Inspection of the PDIP38 sequence shows that there are three putative PCNA-binding motifs. These are shown in alignment (Table I) with those of examples of the known PCNA-binding proteins, p21 (18Gibbs E. Kelman Z. Gulbis J.M. O'Donnell M. Kuriyan J. Burgers P.M.J. Hurwitz J. J. Biol. Chem. 1997; 272: 2373-2381Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 19Warbrick E. Lane D.P. Glover D.M. Cox L.S. Oncogene. 1997; 14: 2313-2321Crossref PubMed Scopus (134) Google Scholar, 20Cox S.L. Trends Cell Biol. 1997; 7: 493-498Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 21Warbrick E. Heatherington W. Lane D.P. Glover D.M. Nucleic Acids Res. 1998; 26: 3925-3933Crossref PubMed Scopus (74) Google Scholar, 28Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar), DNA (cytosine-5)methyltransferase (29Chuang L.S.H. Ian H.I. Koh T.W. Ng H.H. Xu G.L. Li B.F.L. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (779) Google Scholar), the pol δ p68 subunit (11Zhang P. Mo J. Perez A. Leon A. Liu L. Mazloum N. Xu H. Lee M.Y.W.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Hughes P. Tratner I. Ducoux M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar, 13Mo J. Liu L. Rodriquez-Belmonte E.M. Lee M.Y.W.T. Biochemistry. 2000; 39: 7245-7254Crossref PubMed Scopus (49) Google Scholar, 14Liu L. Mo J. Rodriquez-Belmonte E.M. Lee Marietta Y.W.T. J. Biol. Chem. 2000; 275: 18739-18744Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 30Ducoux M. Urbach S. Baldacci G. Hubscher U. Koundrioukoff S. Christensen J. Hughes P. J. Biol. Chem. 2001; 276: 49258-49266Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), FEN1 (19Warbrick E. Lane D.P. Glover D.M. Cox L.S. Oncogene. 1997; 14: 2313-2321Crossref PubMed Scopus (134) Google Scholar), and DNA ligase I (32Levin D.S. Bai W. Yao N. O'Donnell M. Tomkinson A.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12863-12868Crossref PubMed Scopus (193) Google Scholar). The PCNA-binding motif is QXXZXXF(F/Y), where Z is generally an aliphatic residue (20Cox S.L. Trends Cell Biol. 1997; 7: 493-498Abstract Full Text PDF PubMed Scopus (50) Google Scholar). The three candidate sequences (PDIP38) all possess only a single aromatic residue, but there exists at least one known PCNA-binding motif with a single aromatic residue, that for DNA (cytosine-5)methyltransferase (29Chuang L.S.H. Ian H.I. Koh T.W. Ng H.H. Xu G.L. Li B.F.L. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (779) Google Scholar).In yeast, the third subunit of pol δ (10Gerik K.J. Li X. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 31Zuo S. Bermudez V. Zhang G. Kelman Z. Hurwitz J. J. Biol. Chem. 2000; 275: 5153-5162Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) binds to both the homologue of p50 and PCNA. p68, the mammalian third subunit of pol δ, binds to PCNA (11Zhang P. Mo J. Perez A. Leon A. Liu L. Mazloum N. Xu H. Lee M.Y.W.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Hughes P. Tratner I. Ducoux M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar, 13Mo J. Liu L. Rodriquez-Belmonte E.M. Lee M.Y.W.T. Biochemistry. 2000; 39: 7245-7254Crossref PubMed Scopus (49) Google Scholar, 30Ducoux M. Urbach S. Baldacci G. Hubscher U. Koundrioukoff S. Christensen J. Hughes P. J. Biol. Chem. 2001; 276: 49258-49266Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and interacts with the p50 subunit in the yeast two-hybrid assay. 2L. Liu and M. Y. W. T. Lee, unpublished observations. In this respect, the properties of PDIP38 parallel those of the third subunit of pol δ. This similarity raises a possibility that PDIP38 might be able to substitute p68 to form a variant pol δ holoenzyme. This is an intriguing consideration, bearing in mind that pol δ can be regarded as taking part in a number of cellular functions that include (a) synthesis at the leading strand, (b) synthesis at the lagging strand, and (c) DNA repair and recombination processes. Each of these processes may require a specific and likely different assembly of proteins surrounding the pol δ core heterodimer. This issue is also important in considerations of attempts to isolate a pol δ replication complex from tissues or cells, because there may exist different multiprotein complexes involving pol δ. Further studies using the isolated PDIP38 protein are currently underway to determine its ability to interact with the purified pol δ complex and to assess its functional effects on pol δ activity.Although the functions of PDIP38 are at this point unknown, a protein that binds to both p50 and PCNA could serve as a structural role to strengthen the interaction of the pol δ heterodimer with PCNA by binding to pol δ and PCNA, as already noted for p68 (14Liu L. Mo J. Rodriquez-Belmonte E.M. Lee Marietta Y.W.T. J. Biol. Chem. 2000; 275: 18739-18744Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). This possibility is consistent with studies of S. pombe Cdc27, where the three-subunit enzyme in which Cdc27 is absent was found to bind much less strongly to PCNA than did the four-subunit pol δ (31Zuo S. Bermudez V. Zhang G. Kelman Z. Hurwitz J. J. Biol. Chem. 2000; 275: 5153-5162Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Given the trivalent nature of PCNA, there is also the possibility that PDIP38 could serve as a bridging protein between two pol δ-PCNA complexes. This could be envisioned as a linking of two pol δ-PCNA assemblies via PDIP38 in which it is attached to one assembly via PCNA and to the other via p50.Data base analysis showed that PDIP38 has a striking conservation between the C-terminal 111 residues with the bacterial APAG proteins. A similar conservation has been found for one other mammalian protein, the F box A protein (22Ilyin G.P. Rialland M. Pigeon C. Guguen-Guillouzo C. Genomics. 2000; 67: 40-47Crossref PubMed Scopus (56) Google Scholar). We suggest this conserved region in PDIP38 be called the APAG domain. The conservation of the APAG domain across a wide evolutionary range argues that it is likely to have some significant biological function. The functions of the APAG protein or of the members of the family (Fig. 3) that includes PDIP38 are unknown. The presence of the highly conserved GXGXXG motif suggests the presence of a pyrophosphate-binding domain,i.e. that they may be able to bind either pyrophosphate or nucleotide triphosphates (27Aravind L. Koonin E. Nucleic Acids Res. 1998; 26: 3746-3752Crossref PubMed Scopus (196) Google Scholar). Thus, exploration of the possible binding of these compounds to PDIP38 may provide clues to its function. The FBA protein contains an F box at its N terminus (residues 9–58). The latter motif provides the family of F box proteins with the ability to bind to the core of the ubiquitin-protein ligase complex, Skp1-cullin-F box protein (33Elledge S.J. Harper J.W. Biochim. Biophys. Acta. 1998; 1377: M61-M70PubMed Google Scholar). The F box proteins bind to specific substrates, which are thereby targeted for intracellular degradation via the proteosome. The presence of an APAG domain in the F box A protein would suggest that it may be a protein interaction domain; however, the target of the F box A protein is currently unknown.The current work establishes that p50 interacts with PDIP38, and it is noteworthy that p50 has also been reported to interact with a protein termed p36 also known as PDIP1 (polymerase δ interacting protein 1) and the Werner helicase (34He H. Tan C.K. Downey K.M. So A.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11979-11984Crossref PubMed Scopus (47) Google Scholar, 35Szekely A.M. Chen Y.H. Zhang C. Oshima J. Weissman S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11365-11370Crossref PubMed Scopus (102) Google Scholar). This suggests that p50 may serve as a nexus or scaffold for the interaction of other subunits or accessory proteins with pol δ.In summary, we have identified two novel proteins, PDIP38 and PDIP46, that interact with the p50 subunit of pol δ. The interaction of PDIP38 with p50 was extensively characterized, and although its functions are currently unknown, these findings indicate that the number of proteins that may be involved in the formation of the pol δ enzyme complex may involve additional proteins besides the four known subunits. DNA replication is essential not only for duplication of the genome but also for maintenance of genomic integrity during DNA repair (1Hubscher U. Giovanni M. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (584) Google Scholar, 2Sutton M.D. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8342-8349Crossref PubMed Scopus (153) Google Scholar). Chromosomal DNA replication in eukaryotic cells requires three distinct DNA polymerases-α, -δ, and -ε (1Hubscher U. Giovanni M. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (584) Google Scholar, 2Sutton M.D. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8342-8349Crossref PubMed Scopus (153) Google Scholar, 3Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1388) Google Scholar, 4Lee M.Y.W.T. Tan C.-K. Downey K.M. So A.G. Biochemistry. 1984; 23: 1906-1913Crossref PubMed Scopus (174) Google Scholar, 5MacNeill S.A. Moreno S. Reynolds N. Nurse P. Fantes P.A. EMBO J. 1996; 15: 4613-4628Crossref PubMed Scopus (85) Google Scholar, 6Zlotkin T. Kaufmann G. Jiang Y. Lee M.Y.W.T. Uitto L. Syvaojo J. Dornreiter I. Fanning E. Nethanel T. EMBO J. 1996; 15: 2293-2305Crossref Scopus (111) Google Scholar). pol1 δ is required for replication of the leading strand and for completion of the lagging strand synthesis at the replication fork (7Waga S. Stillman B. Nature. 1994; 369: 207-212Crossref PubMed Scopus (493) Google Scholar). The action of pol δ as a processive enzyme requires its interaction with proliferating cell nuclear antigen (PCNA), which functions as a molecular sliding clamp (1Hubscher U. Giovanni M. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (584) Google Scholar). The core mammalian pol δ enzyme consists of a tightly associated heterodimer of 125- and 50-kDa subunits. pol δ has been shown recently (8Zuo S. Gibbs E. Kelven Z. Wang T.S.–F. O'Donnell M. MacNeill S.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11244-11249Crossref PubMed Scopus (70) Google Scholar, 9Reynolds N. Watt A. Fantes P.A. MacNeill S.A. Curr. Genet. 1998; 34: 250-258Crossref PubMed Scopus (50) Google Scholar, 10Gerik K.J. Li X. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 11Zhang P. Mo J. Perez A. Leon A. Liu L. Mazloum N. Xu H. Lee M.Y.W.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Hughes P. Tratner I. Ducoux M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar, 13Mo J. Liu L. Rodriquez-Belmonte E.M. Lee M.Y.W.T. Biochemistry. 2000; 39: 7245-7254Crossref PubMed Scopus (49) Google Scholar, 14Liu L. Mo J. Rodriquez-Belmonte E.M. Lee Marietta Y.W.T. J. Biol. Chem. 2000; 275: 18739-18744Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) to consist of at least four subunits, consisting of the core enzyme and two additional subunits in both yeast and mammalian systems. In the yeast Schizosaccharomyces pombe, Cdc27 and Cdm1 have been identified as the third and the fourth pol δ subunits, respectively, (8Zuo S. Gibbs E. Kelven Z. Wang T.S.–F. O'Donnell M. MacNeill S.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11244-11249Crossref PubMed Scopus (70) Google Scholar, 9Reynolds N. Watt A. Fantes P.A. MacNeill S.A. Curr. Genet. 1998; 34: 250-258Crossref PubMed Scopus (50) Google Scholar). In Saccharomyces cerevisiae Pol32p has been identified as the homologue of the S. pombe third subunit (10Gerik K.J. Li X. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). A human homologue of Cdc27, the KIAA0039 gene product (11Zhang P. Mo J. Perez A. Leon A. Liu L. Mazloum N. Xu H. Lee M.Y.W.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Hughes P. Tratner I. Ducoux M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar, 13Mo J. Liu L. Rodriquez-Belmonte E.M. Lee M.Y.W.T. Biochemistry. 2000; 39: 7245-7254Crossref PubMed Scopus (49) Google Scholar), and p12, a human homologue of Cdm1 (14Liu L. Mo J. Rodriquez-Belmonte E.M. Lee Marietta Y.W.T. J. Biol. Chem. 2000; 275: 18739-18744Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), have recently been identified and can be considered to be the third and fourth subunits of human pol δ. Our laboratory has been interested in the identification of additional protein components that are involved in the formation of the pol δ replication complex. In this study we report the identification of two novel proteins, PDIP38 and PDIP46, that interact with the p50 subunit of pol δ by the use of the yeast two-hybrid (15Fields S. Song O. Nature. 1989; 340: 245-247Crossref PubMed Scopus (4822) Google Scholar,16Hollenberg S.M. Sternganz R. Chang P.-F. Weintraub H. Mol. Cell. Biol. 1995; 5: 3813-3822Crossref Scopus (582) Google Scholar) screening method. PDIP38 was shown to be a PCNA-binding protein, and its interaction with pol δ was established by additional experiments. DISCUSSIONThe motivation for these studies was to identify novel proteins that interact with pol δ using the yeast two-hybrid system. pol δ, rigorously isolated from mammalian tissues, has been shown to consist of a tightly associated heterodimer of the p125 and p50 subunits. Two less tightly associated subunits, p68 and p12, have recently been identified as homologues of the so-called third and fourth subunits inS. pombe (11Zhang P. Mo J. Perez A. Leon A. Liu L. Mazloum N. Xu H. Lee M.Y.W.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Hughes P. Tratner I. Ducoux M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar, 13Mo J. Liu L. Rodriquez-Belmonte E.M. Lee M.Y.W.T. Biochemistry. 2000; 39: 7245-7254Crossref PubMed Scopus (49) Google Scholar, 14Liu L. Mo J. Rodriquez-Belmonte E.M. Lee Marietta Y.W.T. J. Biol. Chem. 2000; 275: 18739-18744Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In S. cerevisiae, evidence for the presence of the third subunit has been obtained, but so far a homologue of the S. pombe fourth subunit (10Gerik K.J. Li X. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) has not been identified. A yeast two-hybrid screen of a human cDNA library using the p125 and p50 subunits was carried out. Our yeast two-hybrid screening using p50 as the bait revealed three positives. Two of these were proteins of unknown function, and the third was identified as p21.Obviously, protein-protein interactions revealed by the yeast two-hybrid system do not necessarily indicate that the native (non-fusion) proteins are capable of interaction, nor do they provide any evidence that such interactions take place in a cellular context. In these studies, we have further characterized the interaction of PDIP38 with the p50 subunit of pol δ.The ability of PDIP38 to interact with p50 and also with PCNA was demonstrated by coimmunoprecipitation and GST pull-down assays. Pull-down experiments using bacterially expressed p50 and PCNA established that the interactions were direct. These experiments were important for the validation of the coimmunoprecipitation experiments from cell extracts. The coimmunoprecipitation experiments revealed that PDIP38 interacts with the pol δ complex in cell extracts, in that we could show the coimmunoprecipitation of p125 as well as p50. The results indicate that PDIP38 is able to bind to p50 when the latter is in association with p125. More significantly, an association of PDIP38 with pol δ activity during immunoaffinity chromatography was established. An association of PDIP38 with pol δ was also demonstrated by native gel electrophoresis of 293 cell lysates. These studies strongly support the view that the interaction of PDIP38 with the pol δ complex is physiological, i.e. that this interaction takes place in a cellular context and is likely to play some physiological relevant function. The issues of whether it is an accessory protein for pol δ or could be considered to be a subunit of pol δ is currently under investigation.Whereas the functions of PDIP38 are still unknown, our studies have shown that it possesses one property that is of relevance to the functions of pol δ, viz. the ability to bind to PCNA. PCNA has been shown to bind a number of proteins through a short peptide motif (18Gibbs E. Kelman Z. Gulbis J.M. O'Donnell M. Kuriyan J. Burgers P.M.J. Hurwitz J. J. Biol. Chem. 1997; 272: 2373-2381Abstract Full Text Full Text PDF PubMed Scopus (51) Googl
Referência(s)