The long-term objective of this project is to understand the underlying mechanisms of mutagenesis and define the mutational pathways and repair systems operating in living cells. Because of the relationship between mutagenesis and carcinogenesis, it is the contention of Dr. Miller that this project has a direct bearing on human health.
The specific aims of the application involve detecting and characterizing different mutator genes in bacteria, and finding and characterizing their homologs in human cDNA and human genomic libraries. Initially, the work will focus on four mutators which were discovered during the previous funding period. The mutY and mutM genes encode proteins that counteract the effects of the oxidation product 8-oxodG. Work is already ongoing with a fragment of the mutY homolog from a human cDNA library, and this fragment will be used to determine the sequence of the entire gene, detect the genomic segment it is carried on, and to map the gene on the human chromosome. Ultimately, it is planned to screen human tumor lines that are mutators for defects in this gene, and others that can be detected which are homologous to bacterial mutators. Additional studies will focus on the mutA and mutC genes which Dr. Miller has found to create mutations by a novel pathway. A missense suppressor is created that inserts glycine at the aspartic acid codon and, in doing so, probably creates a mutator polymerase by substituting in a fraction of the population, a glycine for an essential aspartic acid in the epsilon subunit of polymerase III. It is contended that this pathway of mutagenesis in bacteria has possible implications for understanding contributions to aging and possibly cancer susceptibility in humans.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Chemical Pathology Study Section (CPA)
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University of California Los Angeles
Schools of Medicine
Los Angeles
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Funchain, P; Yeung, A; Stewart, J et al. (2001) Amplification of mutator cells in a population as a result of horizontal transfer. J Bacteriol 183:3737-41
Yang, H; Clendenin, W M; Wong, D et al. (2001) Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch. Nucleic Acids Res 29:743-52
Funchain, P; Yeung, A; Stewart, J L et al. (2000) The consequences of growth of a mutator strain of Escherichia coli as measured by loss of function among multiple gene targets and loss of fitness. Genetics 154:959-70
Slupska, M M; Chiang, J H; Luther, W M et al. (2000) Genes involved in the determination of the rate of inversions at short inverted repeats. Genes Cells 5:425-37
Yang, H; Slupska, M M; Wei, Y F et al. (2000) Cloning and characterization of a new member of the Nudix hydrolases from human and mouse. J Biol Chem 275:8844-53
Miller, J H; Yeung, A; Funchain, P et al. (2000) Temporary and permanent mutators lacking the mismatch repair system: the enhancement of mutators in cell populations. Cold Spring Harb Symp Quant Biol 65:241-52
Miller, J H; Suthar, A; Tai, J et al. (1999) Direct selection for mutators in Escherichia coli. J Bacteriol 181:1576-84
Slupska, M M; Luther, W M; Chiang, J H et al. (1999) Functional expression of hMYH, a human homolog of the Escherichia coli MutY protein. J Bacteriol 181:6210-3
Miller, J H (1998) Mutators in Escherichia coli. Mutat Res 409:99-106
Slupska, M M; King, A G; Lu, L I et al. (1998) Examination of the role of DNA polymerase proofreading in the mutator effect of miscoding tRNAs. J Bacteriol 180:5712-7

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