This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Prostate cancer is the most common malignancy and the second most common cause of cancer death in men in the United States. The molecular etiology of this disease is largely unknown. However, human cancer progresses through the accumulation of somatic mutations. Several years ago it was discovered that mammalian cells need distinct DNA polymerases to efficiently bypass DNA damage without introducing mutations. These polymerases include human pol eta, kappa and beta. Both pol beta and pol kappa are located in chromosomal regions known to be lost during prostate cancer progression, while pol eta mutations cause XPV (Xeroderma pigmentosum-variant), a human disorder characterized by increased predisposition to skin cancer. We present preliminary data that prostate tumors have prevalent somatic missense mutations in all three genes. Our long-term goal is to characterize the contribution of pol beta, eta and kappa to prostate cancer predisposition and progression. In this proposal we plan: (1) to identify common somatic variants in the human pol beta, eta and kappa genes by sequencing the entire coding region of these genes using microdissected prostate cancer tissue and matched constitutional DNA from 30 prostate cancer patients. (2) To measure the frequency and distribution by tumor stage, tumor grade and patient age of each of the variants identified above in DNA isolated from 200 prostate cancer tissues, by genotyping with the multiplex SNaPshot kit. (3). to construct the polymerase variants identified above in appropriate cDNA expression vectors and purify them by columns. Following purification, we will biochemically characterize each purified polymerase variant by measuring their respective kinetic parameters compared to wild type by enzyme assays, using appropriate DNA substrates. This analysis will allow us to assess the functional effects of these variants on activity and fidelity of DNA replication.
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