{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,25]],"date-time":"2026-04-25T03:34:32Z","timestamp":1777088072928,"version":"3.51.4"},"reference-count":51,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5334","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1997,9,26]]},"abstract":"<jats:p>\n            DNA\u2013(cytosine-5) methyltransferase (MCMT) methylates newly replicated mammalian DNA, but the factors regulating this activity are unknown. Here, MCMT is shown to bind proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA replication and repair. Binding of PCNA requires amino acids 163 to 174 of MCMT, occurs in intact cells at foci of newly replicated DNA, and does not alter MCMT activity. A peptide derived from the cell cycle regulator p21\n            <jats:sup>WAF1<\/jats:sup>\n            can disrupt the MCMT-PCNA interaction, which suggests that p21\n            <jats:sup>WAF1<\/jats:sup>\n            may regulate methylation by blocking access of MCMT to PCNA. MCMT and p21\n            <jats:sup>WAF1<\/jats:sup>\n            may be linked in a regulatory pathway, because the extents of their expression are inversely related in both SV40-transformed and nontransformed cells.\n          <\/jats:p>","DOI":"10.1126\/science.277.5334.1996","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:44:47Z","timestamp":1027763087000},"page":"1996-2000","source":"Crossref","is-referenced-by-count":731,"title":["Human DNA-(Cytosine-5) Methyltransferase-PCNA Complex as a Target for p21\n            <sup>WAF1<\/sup>"],"prefix":"10.1126","volume":"277","author":[{"given":"Linda S.-H.","family":"Chuang","sequence":"first","affiliation":[{"name":"L. S.-H. Chuang, H.-I. Ian, T.-W. Koh, H.-H. Ng, B. F. L. Li, Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."},{"name":"G. Xu, Bioscience Centre, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."}]},{"given":"Hang-In","family":"Ian","sequence":"additional","affiliation":[{"name":"L. S.-H. Chuang, H.-I. Ian, T.-W. Koh, H.-H. Ng, B. F. L. Li, Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."},{"name":"G. Xu, Bioscience Centre, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."}]},{"given":"Tong-Wey","family":"Koh","sequence":"additional","affiliation":[{"name":"L. S.-H. Chuang, H.-I. Ian, T.-W. Koh, H.-H. Ng, B. F. L. Li, Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."},{"name":"G. Xu, Bioscience Centre, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."}]},{"given":"Huck-Hui","family":"Ng","sequence":"additional","affiliation":[{"name":"L. S.-H. Chuang, H.-I. Ian, T.-W. Koh, H.-H. Ng, B. F. L. Li, Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."},{"name":"G. Xu, Bioscience Centre, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."}]},{"given":"Guoliang","family":"Xu","sequence":"additional","affiliation":[{"name":"L. S.-H. Chuang, H.-I. Ian, T.-W. Koh, H.-H. Ng, B. F. L. Li, Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."},{"name":"G. Xu, Bioscience Centre, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."}]},{"given":"Benjamin F. L.","family":"Li","sequence":"additional","affiliation":[{"name":"L. S.-H. Chuang, H.-I. Ian, T.-W. Koh, H.-H. Ng, B. F. L. Li, Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."},{"name":"G. Xu, Bioscience Centre, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Republic of Singapore."}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/366362a0"},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1016\/S0092-8674(00)81887-5","volume":"88","author":"Nan X.","year":"1997","unstructured":"Nan X., Campoy F. 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A., Cancer Res. 56, 2463 (1996);","journal-title":"Cancer Res."},{"key":"e_1_3_1_5_5","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.94.6.2545"},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1146\/annurev.ne.18.030195.000453","volume":"18","author":"Warrent S. T.","year":"1995","unstructured":"Warrent S. T., Ashley C. T., Annu. Rev. Neurosci. 18, 77 (1995).","journal-title":"Annu. Rev. Neurosci."},{"key":"e_1_3_1_7_2","unstructured":"Abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr."},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1016\/0092-8674(92)90561-P","volume":"71","author":"Leonhardt H.","year":"1992","unstructured":"Leonhardt H., Page A. W., Weier H. U., Bestor T. 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K., et al., Biochemistry 35, 12259 (1996).","journal-title":"Biochemistry"},{"key":"e_1_3_1_13_2","doi-asserted-by":"crossref","first-page":"3195","DOI":"10.1242\/dev.122.10.3195","volume":"122","author":"Lei H.","year":"1996","unstructured":"Lei H., et al., Development 122, 3195 (1996).","journal-title":"Development"},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","first-page":"1232","DOI":"10.1016\/S0960-9822(95)00245-4","volume":"5","author":"Krude T.","year":"1995","unstructured":"Krude T., Curr. Biol. 5, 1232 (1995);","journal-title":"Curr. 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O'Donnell"},{"key":"e_1_3_1_15_3","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1016\/0092-8674(92)90445-I","volume":"69","author":"Kuriyan J.","year":"1992","unstructured":"Kuriyan J., Cell 69, 425 (1992);","journal-title":"Cell"},{"key":"e_1_3_1_15_4","first-page":"1233","volume":"79","author":"Krishna T. S. R.","year":"1994","unstructured":"Krishna T. S. R., Kong X.-P., Gary S., Burgers P. M., Kuriyan J., ibid. 79, 1233 (1994);","journal-title":"ibid."},{"key":"e_1_3_1_15_5","doi-asserted-by":"crossref","unstructured":"; D. R. Herendeen and T. J. Kelly ibid. 84 5 (1996); V. Naktinis J. Turner M. O\u2032Donnell ibid. p. 137.","DOI":"10.1016\/S0092-8674(00)81000-4"},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1006\/jmbi.1996.0213","volume":"257","author":"Chuang L. S.-H.","year":"1996","unstructured":"Chuang L. S.-H., Ng H.-H., Chia J. N., Li B. F. L., J. Mol. Biol. 257, 935 (1996).","journal-title":"J. Mol. 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Acad. Sci. U.S.A."},{"key":"e_1_3_1_18_3","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(17)35981-1"},{"key":"e_1_3_1_18_4","doi-asserted-by":"crossref","first-page":"1235","DOI":"10.1093\/jnci\/85.15.1235","volume":"85","author":"Issa J. P.","year":"1993","unstructured":"Issa J. P., et al., J. Natl. Cancer Inst. 85, 1235 (1993).","journal-title":"J. Natl. Cancer Inst."},{"key":"e_1_3_1_19_2","unstructured":"A. K. C. Teo and B. F. L. Li unpublished data."},{"key":"e_1_3_1_20_2","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1016\/0092-8674(92)90416-A","volume":"69","author":"Shivji M. K.","year":"1992","unstructured":"Shivji M. K., Kenny M. K., Wood R. D., Cell 69, 367 (1992);","journal-title":"Cell"},{"key":"e_1_3_1_20_3","doi-asserted-by":"crossref","first-page":"2441","DOI":"10.1093\/nar\/20.10.2441","volume":"20","author":"Nichols A.","year":"1992","unstructured":"Nichols A., Sancar A., Nucleic Acids Res. 20, 2441 (1992);","journal-title":"Nucleic Acids Res."},{"key":"e_1_3_1_20_4","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/S0092-8674(00)81323-9","volume":"87","author":"Umar A.","year":"1996","unstructured":"Umar A., et al., Cell 87, 65 (1996).","journal-title":"Cell"},{"key":"e_1_3_1_21_2","doi-asserted-by":"crossref","first-page":"1062","DOI":"10.1016\/S0960-9822(00)00244-X","volume":"4","author":"Shivji M. K.","year":"1994","unstructured":"Shivji M. K., Grey S. J., Strausfeld U. P., Wood R. D., Blow J. J., Curr. Biol. 4, 1062 (1994);","journal-title":"Curr. Biol."},{"key":"e_1_3_1_21_3","doi-asserted-by":"crossref","first-page":"574","DOI":"10.1038\/369574a0","volume":"369","author":"Waga S.","year":"1994","unstructured":"Waga S., Hannon G. J., Beach D., Stillman B., Nature 369, 574 (1994);","journal-title":"Nature"},{"key":"e_1_3_1_21_4","first-page":"159","volume":"375","author":"Luo Y.","year":"1995","unstructured":"Luo Y., Hurwitz J., Massague J., ibid. 375, 159 (1995).","journal-title":"ibid."},{"key":"e_1_3_1_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(93)90500-P"},{"key":"e_1_3_1_23_2","first-page":"6423","volume":"52","author":"Ayi T.-C.","year":"1992","unstructured":"Ayi T.-C., Loh K.-C., Ali R. B., Li B. F. L., Cancer Res. 52, 6423 (1992);","journal-title":"Cancer Res."},{"key":"e_1_3_1_23_3","doi-asserted-by":"crossref","first-page":"4050","DOI":"10.1002\/j.1460-2075.1996.tb00778.x","volume":"15","author":"Lim A.","year":"1996","unstructured":"Lim A., Li B. F. L., EMBO J. 15, 4050 (1996);","journal-title":"EMBO J."},{"key":"e_1_3_1_23_4","unstructured":"; J. Sambrook E. F. Fritsch T. Maniatis Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1989)."},{"key":"e_1_3_1_24_2","unstructured":"The cloning and expression procedures were described previously for F122\u20131616 and F323\u20131616 fragments (F) of MCMT (15). F122\u2013418 in pGEX1was an Eco RI\u2013Bst EII fragment. F6 in pGEX3 and F5 in pGEX1 were Mae II fragments of F122\u2013418. F3 and F4 in pGEX2T were obtained with the polymerase chain reaction (PCR). F1 F2 and all vertebrate MPBD derivatives were cloned by oligonucleotide duplexes with Eco RI and Bam HI overhangs into pGEX2T (15). Human PCNA was cloned by PCR into pET3a. All sequences were confirmed by dideoxy sequencing. Proteins were induced with either 5 \u03bcM isopropyl-\u03b2- d -thiogalactoside (IPTG) for 48 hours at 22\u00b0C or 0.1 to 1 mM IPTG at 37\u00b0C for 3 hours. Proteins from bacterial lysates (15) were either purified on GSH-Sepharose or precipitated with ammonium sulfate before further purification."},{"key":"e_1_3_1_25_2","unstructured":"For immunochemistry cell extracts were prepared as described (11). Antibodies used are as follows: PC10 (anti-PCNA) from Santa Cruz; anti-GST Protein-G \u03b3 purified rabbit antibodies raised against GST from pGEX2T; mAb D12 raised against GST-(F323\u20131616); anti-MCMT GST-(F323\u20131616) affinity column\u2013 purified rabbit antibodies raised against GST(F122\u20131616); mAb Cip1 (anti-p21 WAF1 ) from Transduction Lab. For immunoprecipitation cell extract (2 mg) was incubated with anti-MCMT (13 \u03bcg) in IP buffer [equal volume of 50 mM tris (pH 7.5) 5% glycerol and 0.2% Triton X-100] at 4\u00b0C for 1 hour. Protein G \u03b3 \u2013Sepharose (60 \u03bcl) beads were added for 2 hours. After washing three times with 500 \u03bcl of IP buffer (with 0.1 M NaCl) samples were boiled in Laemmli buffer for immunoblot."},{"key":"e_1_3_1_26_2","unstructured":"For PCNA-binding CEM cell extract (11) or rPCNA were added to binding buffer [100 \u03bcl 50 mM tris (pH 7.5) 0.1% Triton X-100 28 \u03bcM ZnCl 2 10% glycerol and 0.22 M KCl)] with GSH\u2013Sepharose bead\u2013bound fusion proteins. After shaking at 4\u00b0C for 40 min the beads were recovered and washed three times with 500 \u03bcl of binding buffer before boiling in Laemmli buffer for immunoblot with PC10 antibody. In peptide competition assays high-performance liquid chromatography (HPLC)\u2013purified dodecapeptides (Research Genetics) were reconstituted to 2 mg\/ml with argon-treated water and used directly."},{"key":"e_1_3_1_27_2","unstructured":"For transient transfection assay (22) the WT GST-(F122\u2013207) construct was obtained from F122\u20131616 by PCR and cloned into Not I\u2013Kpn I sites of pXJ41neo. This was used for site-directed mutagenesis to create the H170V mutant (confirmed by dideoxy sequencing). After transfection MRC5SV cells were labeled with BrdU (cell proliferation kit Amersham) before staining (7 22)."},{"key":"e_1_3_1_28_2","unstructured":"Methylase activity assays were performed as described (9) but at 25\u00b0C with 10 min preincubation at 37\u00b0C. Aliquots (100 \u03bcl) were analyzed for tritiated poly(dI-dC) at 10-min intervals."},{"key":"e_1_3_1_29_2","unstructured":"For [ 3 H]SAM labeling cells in a 175-cm 2 (surface area) flask (75% confluent) were treated with 0 6 -benzylguanine [17 ml of 10 \u03bcM in serum-free media (SFM)] for 1 hour followed by addition of NMU (1 ml of 2.7 mM in SFM) or SFM with dimethyl sulfoxide as control (11). After 40 min the media were removed and 11 ml of labeling mixture containing 1.1 ml of dialyzed fetal bovine serum and 100 \u03bcCi of [ 3 H]SAM (75 Ci\/mmol Amersham) in amino acid\u2013free modified Eagle's medium was added for 6 or 16 hours. Genomic DNAs were isolated as described (22) and digested with venom phosphodiesterase (15 U) and shrimp alkaline phosphatase (10 U) in digestion buffer [25 mM tris (pH 8.0) with 2.5 mM Mg 2+ 300 \u03bcl per flask] for 16 hours at 37\u00b0C. The deoxynucleosides were analyzed by HPLC on a C-18 reversed-phase cartridge as described (9) and were quantified using standard 2\u2032-deoxynucleosides (Sigma). [ 3 H]5meC was determined by scintillation counting of 0.5-ml fractions collected from each HPLC run and normalized to 1 mmol of total nucleosides detected. Two independent labeling experiments were performed."},{"key":"e_1_3_1_30_2","unstructured":"We thank E. Manser for critical reading of the manuscript; Y. H. Tan and T. J. Lam for stimulating this collaboration; and R. B. Ali A. Teo and H. K. Oh for excellent assistance. Supported by the National Science and Technology Board of Singapore. This paper is dedicated to P. F. Swann (University College London) for his constant encouragement to B.F.L.L."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.277.5334.1996","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T03:52:21Z","timestamp":1705117941000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.277.5334.1996"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1997,9,26]]},"references-count":51,"journal-issue":{"issue":"5334","published-print":{"date-parts":[[1997,9,26]]}},"alternative-id":["10.1126\/science.277.5334.1996"],"URL":"https:\/\/doi.org\/10.1126\/science.277.5334.1996","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1997,9,26]]}}}