Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21(WAF1)

Science

Volumn 277, Issue 5334, 1997, Pages 1996-2000

  Chuang, Linda S H ^a   Ian, Hang In ^a   Koh, Tong Wey ^a   Ng, Huck Hui ^a   Xu, Guoliang ^a   Li, Benjamin F L ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

CYCLINE; DNA (CYTOSINE 5) METHYLTRANSFERASE; PROTEIN P21; RECOMBINANT PROTEIN;

ARTICLE; CELL CYCLE; DNA REPLICATION; HUMAN; HUMAN CELL; PRIORITY JOURNAL; PROTEIN BINDING; PROTEIN EXPRESSION; PROTEIN METHYLATION; PROTEIN PROTEIN INTERACTION; SIMIAN VIRUS 40; VIRUS CELL TRANSFORMATION;

AMINO ACID SEQUENCE; CELL LINE, TRANSFORMED; CELL NUCLEUS; CYCLIN-DEPENDENT KINASE INHIBITOR P21; CYCLINS; DNA (CYTOSINE-5-)-METHYLTRANSFERASE; DNA DAMAGE; DNA METHYLATION; DNA REPAIR; DNA REPLICATION; HUMANS; MOLECULAR SEQUENCE DATA; PEPTIDES; PROLIFERATING CELL NUCLEAR ANTIGEN; RECOMBINANT FUSION PROTEINS; TUMOR CELLS, CULTURED;

EID: 0030770835     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5334.1996     Document Type: Article

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45. The cloning and expression procedures were described previously for F122-1616 and F323-1616 fragments (F) of MCMT (15). F122-418 in pGEX1was an Eco RI-Bst Ell fragment. F6 in pGEX3 and F5 in pGEX1 were Mae II fragments of F122-418. 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 μM isopropyl-β-D-thiogalactoside (IPTG) for 48 hours at 22°C or 0.1 to 1 mM IPTG at 37°C for 3 hours. Proteins from bacterial lysates (15) were either purified on GSH-Sepharose or precipitated with ammonium sulfate before further purification.
46. γ-Sepharose (60 μl) beads were added for 2 hours. After washing three times with 500 μl of IP buffer (with 0.1 M NaCl), samples were boiled in Laemmli buffer for immunoblot.
47. 2, 10% glycerol, and 0.22 M KCI)] with GSH-Sepharose bead-bound fusion proteins. After shaking at 4°C for 40 min, the beads were recovered and washed three times with 500 μl of binding buffer before boiling in Laemmli buffer for immunoblot with PC10 antibody. In peptide competition assays, high-performance liquid chromatography (HPLC)-purified dodecapeptides (Research Genetics) were reconstituted to 2 mg/ml with argon-treated water and used directly.
48. For transient transfection assay (22), the WT GST-(F122-207) construct was obtained from F122-1616 by PCR and cloned into Not I-Kpn I sites of pXJ41 neo. 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).
49. Methylase activity assays were performed as described (9) but at 25°C with 10 min preincubation at 37°C. Aliquots (100 μl) were analyzed for tritiated poly(dl-dC) at 10-min intervals.
50. 3H]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.
51. 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.