Abstract

The long-term objectives of this application are to obtain a detailed understanding of the structure, mechanisms of action, and regulation of the human DNA methyltransferase(s) (E.C.2.1.1.37). Available evidence strongly suggests that the postsynthetic modification of DNA catalyzed by this enzyme has a role in cellular differentiation and somatic inheritance. DNA methylation may also be involved in ectopic protein production, tumor initiation, and tumor progression. We propose to prepare and study milligram quantitites of the homogeneous enzyme from human placenta. Experiments designed to study the enzyme structure include the determination of sub-unit stoichiometry and molecular weight, and a partial determination of amino acid sequence. The purified enzyme will be used to construct and optimize an in vitro DNA methylation system. This system and antisera to the purified enzyme will be used in experiments to identify factors that may promote or inhibit DNA methylation during cell proliferation, and factors that may be responsible for the hypomethylation of DNA observed in certain transformed cell lines. The amino acid sequence information will be used to synthesize oligonucleotides for use as hybridization probes to aid in the isolation of a cDNA clone for the methyltransferase. The cDNA clone(s) thus isolated will be used to determine the genomic organization and the transcriptional regulation of the methyltransferase gene(s).

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM032863-03S1
Application #
3282044
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-12-01
Project End
1988-09-25
Budget Start
1987-08-01
Budget End
1988-09-25
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
City
Duarte
State
CA
Country
United States
Zip Code
91010

  Publications

Clark, Jarrod; Smith, Steven S (2008) Secondary structure at a hot spot for DNA methylation in DNA from human breast cancers. Cancer Genomics Proteomics 5:241-51
Woodcock, D M; Crowther, P J; Diver, W P et al. (1988) RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase. Nucleic Acids Res 16:4465-82
Heby, O; Persson, L; Smith, S S (1988) Polyamines, DNA methylation and cell differentiation. Adv Exp Med Biol 250:291-9
Benfield, J R; Wain, J C; Derrick, M et al. (1988) Biochemical and cytogenetic studies of human lung cancers. J Thorac Cardiovasc Surg 96:840-8
Smith, S S; Hardy, T A; Baker, D J (1987) Human DNA (cytosine-5)methyltransferase selectively methylates duplex DNA containing mispairs. Nucleic Acids Res 15:6899-916
Tolberg, M E; Funderburk, S J; Klisak, I et al. (1987) Structural organization and DNA methylation patterning within the mouse L1 family. J Biol Chem 262:11167-75
Baker, D J; Hardy, T A; Smith, S S (1987) The influence of the dT.dG mispair on the activity of the human DNA(cytosine-5)methyltransferase. Biochem Biophys Res Commun 146:596-602
Hardy, T A; Baker, D J; Newman, E M et al. (1987) Size of the directing moiety at carbon 5 of cytosine and the activity of human DNA(cytosine-5) methyltransferase. Biochem Biophys Res Commun 145:146-52
Wain, J C; Wilkins, S J; Benfield, J R et al. (1986) Altered DNA methylation patterns in human lung carcinomas. Curr Surg 43:489-92
DePaoli-Roach, A; Roach, P J; Zucker, K E et al. (1986) Selective phosphorylation of human DNA methyltransferase by protein kinase C. FEBS Lett 197:149-53

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