The DNA adenine methylase, specified by the dam gene of E. coli, has a role in spontaneous mutability. Spontaneous mutability increases substantially if the enzyme is absent or if it is overproduced. This poses two questions: (i) How is the dam gene regulated?, and (ii) What is the mutation spectrum in cells overproducing the enzyme or in which it is not produced at all? This proposal seeks to answer these questions through a combined in vitro and in vivo approach. In vitro manipulations and mutations generated in vivo will be used to identify the promoter(s) and other regulatory elements which control the expression of the dam gene. In vivo generated mutations in a small repressor gene will be analyzed by in vitro DNA sequencing to identify mutations at the nucleotide level. An analysis of the spectrum will test the model that methylation dependent mismatch repair is a major mechanism for removal of potential frameshift mutations. This proposal also seeks to determine which genes of E. coli can have their expression altered by DNA methylation. This will be done by examing expression of fused genes in the presence or absence of DNA adenine methylase. The studies with DNA adenine methylase function in DNA repair and gene expression in the E. coli model systems offers a new approach to the elucidation of the origin and maintenance of mutations and, thus, may contribute significantly to a more thorough understanding of the process linking carcinogenesis and spontaneous congenital abnormalities in man.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030330-06
Application #
3278020
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1982-04-01
Project End
1988-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
6
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Boye, E; Marinus, M G; Lobner-Olesen, A (1992) Quantitation of Dam methyltransferase in Escherichia coli. J Bacteriol 174:1682-5
Wu, T H; Grelland, E; Boye, E et al. (1992) Identification of a weak promoter for the dam gene of Escherichia coli. Biochim Biophys Acta 1131:47-52
Parker, B; Marinus, M G (1988) A simple and rapid method to obtain substitution mutations in Escherichia coli: isolation of a dam deletion/insertion mutation. Gene 73:531-5
Barras, F; Marinus, M G (1988) Arrangement of Dam methylation sites (GATC) in the Escherichia coli chromosome. Nucleic Acids Res 16:9821-38
Marinus, M G (1987) DNA methylation in Escherichia coli. Annu Rev Genet 21:113-31
Schlagman, S L; Hattman, S; Marinus, M G (1986) Direct role of the Escherichia coli Dam DNA methyltransferase in methylation-directed mismatch repair. J Bacteriol 165:896-900
Fram, R J; Cusick, P S; Marinus, M G (1986) Studies on mutagenesis and repair induced by platinum analogs. Mutat Res 173:13-8
Marinus, M G (1985) DNA methylation influences trpR promoter activity in Escherichia coli K-12. Mol Gen Genet 200:185-6
Peterson, K R; Wertman, K F; Mount, D W et al. (1985) Viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants requires increased expression of specific genes in the SOS regulon. Mol Gen Genet 201:14-9
Fram, R J; Cusick, P S; Wilson, J M et al. (1985) Mismatch repair of cis-diamminedichloroplatinum(II)-induced DNA damage. Mol Pharmacol 28:51-5