MUTATIONAL MECHANISMS OF REPETITIVE DNA IN HUMAN CELLS

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The goal of this research proposal is to evaluate the significance of spontaneous and carcinogen-induced mutations at short tandem repeat (STR) DNA loci in human cells. We propose to utilize complementary in vitro/ex vivo mutagenesis assays to test the hypothesis that DNA polymerase errors are a potential source of mutations observed at STR loci in vivo. Bimodal target sequence containing an STR motif and a unique sequence motif will be constructed by inserting dinucleotide and tetranucleotide STR sequences in-frame within the 5 prime coding region of the Herpes simplex virus thymidine kinase (HSV-tk) gene. DNA sequences that make up the ATP-binding site constitute the unique DNA control for which to compare directly the frequency of errors at the STR motif. These targets will be used as DNA templates during in vitro DNA synthesis catalyzed by human DNA polymerase beta and the calf thymus polymerase alpha-primase complex. The mutations produced will be analyzed to quantitate the relative frequencies of polymerase-mediated errors in the two motifs and to ascertain precise in vitro polymerase error rates in STR sequences as a function of repeat size and base composition. The bimodal targets sequences also will be incorporated into ori-rho-tk shuttle vectors which replicate episomally in human lymphoblastoid cells. Mutations produced during replication of the shuttle vectors under defined conditions of leading and lagging strand DNA replication in culture normal cells will be analyzed to determine quantitative mutation rates for STR loci as a function of sequence composition. The bimodal shuttle vectors will be used to demonstrate whether DNA adducts produced by two distinct carcinogens, the arylaminating agent N- benzoyloxy-N-methyl-4- aminoazobenzene and the alkylating agent N- ethyl-N-nitrosourea, can induce mutations in the STR motifs. Two pathways for mutation induction will be followed, the gain/loss of integral repeat units in continuous repeat arrays and base substitution mutations in discontinuous repeat arrays. Data generated by this proposal will: I) enhance our understanding of the mechanisms of human somatic cell mutagenesis by providing quantitation of mutation rates in repetitive DNA, and II) establish the degree to which repetitive DNA is destabilized by chemical carcinogens. Our long-term research objective is to test the hypothesis that mutations in repetitive DNA provide an important source of genotypic variation that drives neoplastic progression.