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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA073649-04
Application #
6150060
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Okano, Paul
Project Start
1997-04-05
Project End
2002-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
4
Fiscal Year
2000
Total Cost
$140,155
Indirect Cost
Name
Pennsylvania State University
Department
Pathology
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
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Maneval, Mandy L; Eckert, Kristin A (2004) Effects of oxidative and alkylating damage on microsatellite instability in nontumorigenic human cells. Mutat Res 546:29-38
Eckert, Kristin A; Mowery, Andrew; Hile, Suzanne E (2002) Misalignment-mediated DNA polymerase beta mutations: comparison of microsatellite and frame-shift error rates using a forward mutation assay. Biochemistry 41:10490-8
Eckert, Kristin A; Yan, Guang; Hile, Suzanne E (2002) Mutation rate and specificity analysis of tetranucleotide microsatellite DNA alleles in somatic human cells. Mol Carcinog 34:140-50
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Eckert, K A; Yan, G (2000) Mutational analyses of dinucleotide and tetranucleotide microsatellites in Escherichia coli: influence of sequence on expansion mutagenesis. Nucleic Acids Res 28:2831-8
Hile, S E; Yan, G; Eckert, K A (2000) Somatic mutation rates and specificities at TC/AG and GT/CA microsatellite sequences in nontumorigenic human lymphoblastoid cells. Cancer Res 60:1698-703