MUTATIONAL MECHANISMS OF REPETITIVE DNA IN HUMAN CELLS

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date4/5/971/31/03