Human DNA-(Cytosine-5) Methyltransferase-PCNA Complex as a Target for p21^WAF1

Li E., Beard C., Jaenisch R., Nature 366, 362 (1993).

Nan X., Campoy F. J., Bird A., Cell 88, 471 (1997).

Li E., Bestor T. H., Jaenisch R., ibid. 69, 915 (1992).

Baylin S. B., et al., Cancer Cells 3, 383 (1991);

Counts J. L., Goodman J. I., Cell 83, 13 (1995);

Jones P. A., Cancer Res. 56, 2463 (1996);

Lengauer C., Kinzler K. W., Vogelstein B., Proc. Natl. Acad. Sci. U.S.A. 94, 2545 (1997).

Warrent S. T., Ashley C. T., Annu. Rev. Neurosci. 18, 77 (1995).

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.

Leonhardt H., Page A. W., Weier H. U., Bestor T. H., Cell 71, 865 (1992).

Chen Y. Q., Cipriano S. C., Arenkiel J. M., Miller F. R., Cancer Res. 55, 4536 (1995);

Warbrick E., Lane D. P., Glover D. M., Cox L. S., Curr. Biol. 5, 275 (1995);

Gulbis J. M., et al., Cell 87, 297 (1996).

Tan N. W., Li B. F. L., Biochemistry 29, 9234 (1990).

Toschi L., Bravo R., J. Cell Biol. 107, 1623 (1988);

Li R., Hannon G. J., Beach D., Stillman B., Curr. Biol. 6, 189 (1996).

Oh H. K., et al., Biochemistry 35, 12259 (1996).

Lei H., et al., Development 122, 3195 (1996).

Krude T., Curr. Biol. 5, 1232 (1995);

Gasser R., Koller T., Sogo J. M., J. Mol. Biol. 258, 224 (1996);

Carotti D., Funiciello S., Lavia P., Caiafa P., Strom R., Biochemistry 35, 11660 (1996);

McArthur M., Thomas J. O., EMBO J. 15, 1705 (1996).

X.-P. Kong, R. Onrust, M. O'Donnell,

Kuriyan J., Cell 69, 425 (1992);

Krishna T. S. R., Kong X.-P., Gary S., Burgers P. M., Kuriyan J., ibid. 79, 1233 (1994);

; D. R. Herendeen and T. J. Kelly, ibid.84, 5 (1996); V. Naktinis, J. Turner, M. O′Donnell, ibid., p. 137.

Chuang L. S.-H., Ng H.-H., Chia J. N., Li B. F. L., J. Mol. Biol. 257, 935 (1996).

Xiong Y., Zhang H., Beach D., Genes Dev. 7, 1572 (1993);

Peterson S. R., Gadbois D. M., Bradbury E. M., Kraemer P. M., Cancer Res. 55, 4651 (1995);

Xiong Y., et al., J. Virol. 70, 999 (1996).

Wu J., et al., Proc. Natl. Acad. Sci. U.S.A. 90, 8891 (1993);

Kautiainen T. L., Jones P. A., J. Biol. Chem. 261, 1594 (1986);

Issa J. P., et al., J. Natl. Cancer Inst. 85, 1235 (1993).

A. K. C. Teo and B. F. L. Li, unpublished data.

Shivji M. K., Kenny M. K., Wood R. D., Cell 69, 367 (1992);

Nichols A., Sancar A., Nucleic Acids Res. 20, 2441 (1992);

Umar A., et al., Cell 87, 65 (1996).

Shivji M. K., Grey S. J., Strausfeld U. P., Wood R. D., Blow J. J., Curr. Biol. 4, 1062 (1994);

Waga S., Hannon G. J., Beach D., Stillman B., Nature 369, 574 (1994);

Luo Y., Hurwitz J., Massague J., ibid. 375, 159 (1995).

El-Deiry W. S., et al., Cell 75, 817 (1993).

Ayi T.-C., Loh K.-C., Ali R. B., Li B. F. L., Cancer Res. 52, 6423 (1992);

Lim A., Li B. F. L., EMBO J. 15, 4050 (1996);

; J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

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 EII 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.

For immunochemistry, cell extracts were prepared as described (11). Antibodies used are as follows: PC10 (anti-PCNA) from Santa Cruz; anti-GST, Protein-G^γ purified rabbit antibodies raised against GST from pGEX2T; mAb D12 raised against GST-(F323–1616); anti-MCMT, GST-(F323–1616) affinity column– purified rabbit antibodies raised against GST(F122–1616); mAb Cip1 (anti-p21^WAF1), from Transduction Lab. For immunoprecipitation, cell extract (2 mg) was incubated with anti-MCMT (13 μg) in IP buffer [equal volume of 50 mM tris (pH 7.5), 5% glycerol, and 0.2% Triton X-100] at 4°C for 1 hour. Protein G^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.

For PCNA-binding, CEM cell extract (11) or rPCNA were added to binding buffer [100 μl, 50 mM tris (pH 7.5), 0.1% Triton X-100, 28 μM ZnCl₂, 10% glycerol, and 0.22 M KCl)] 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.

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 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).

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(dI-dC) at 10-min intervals.

For [³H]SAM labeling, cells in a 175-cm² (surface area) flask (75% confluent) were treated with 0⁶-benzylguanine [17 ml of 10 μM 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 μCi of [³H]SAM (75 Ci/mmol, Amersham) in amino acid–free 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 Mg²⁺, 300 μl per flask] for 16 hours at 37°C. The deoxynucleosides were analyzed by HPLC on a C-18 reversed-phase cartridge as described (9) and were quantified using standard 2′-deoxynucleosides (Sigma). [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.

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.