The long-term goal of our research is to elucidate the role of microsatellite DNA sequence variation in neoplastic progression. The objective of this proposal is to identify the full complement of biochemical mechanisms that act to stabilize microsatellite sequences in human cells. Our working hypothesis is that cellular microsatellite mutation rates are the cumulative result of proteins acting to maintain genomic stability during DNA replication. We have developed complementary in vitro/ex vivo assays to study mutagenesis within reporter microsatellites in somatic human cells.
Specific Aim 1 will test the hypothesis that DNA polymerase pausing within microsatellite sequences can impede replication fork progression, and that RecQ helicases have a specialized function during microsatellite DNA replication. Biochemical analyses of replication intermediates through microsatellites of differing sequence will be performed using cell lines from normal, Bloom and Werner syndrome donors to the function of BLM and WRN helicases. Mutation rates within the herpes simplex virus thymidine kinase (HSV-tk) gene reporter cassettes will be quantitated to determine whether these helicases function to stabilize microsatellite DNA sequences.
Specific Aim 2 will determine the contribution of enzymatic activities associated with the replication fork in maintaining genome stability. We will test the contribution of mismatch repair proteins to the stability of tetranucleotide alleles and microsatellites with potential secondary structure, and test the contribution of the Mre11/NBS/Rad50 complex to human cell replication fidelity. The ex vivo shuttle vector system will be used in naturally occurring MLH1, PMS2, NBS1 and hMre11-defective lymphoblastoid cell lines, and in cells with gene expression down-regulated by antisense methods. Mutation rates and specificities will be determined to establish whether the activities of NBS and hMre11 affect replication fidelity.
Specific Aim 3 will determine the relative contribution of replicative and Y family DNA polymerases to spontaneous mutagenesis and microsatellite stability. The in vitro HSV-tk assay will be used to analyze DNA polymerase delta and polymerase kappa (pol kappa) error rates at microsatellites. The effects of pol kappa levels on spontaneous cellular mutagenesis will be analyzed using the ex vivo assay cell lines containing either pol kappa overexpression vectors or stable ribozymes to down-regulated pol kappa expression. These studies will establish whether regulation of pol kappa activity is a potential avenue for therapeutic interventions aimed at regulating genome stability. This proposed research has direct implications for modeling tumor progression, as the loss of genomic surveillance mechanisms will accelerate microsatellite mutagenesis. Microsatellite allele lengths can directly affect gene expression. As microsatellites are polymorphic in human populations, this effect on gene regulation may be an important factor contributing to individual cancer risk.
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