REPLICON MISFUNCTIONS IN HUMAN CARCINOGENESIS

说明：致癌物可以瞬时或稳定地破坏DNA

DESCRIPTION: Carcinogenic agents that damage DNA may transiently or stably disrupt the highly organized process of DNA replication at individual replicons and lead to mutations. The goals of this application center on the identifying and understanding the molecular factors in human cells involved in bypass replication of cyclobotane thymine-thymine dimers (T=T). DNA replication in cell-free extracts will be analyzed on defined episomal DNA containing a unique T=T dimer. Two focused specific aims were described to achieve these objectives. The first aim seeks to determine the molecular defect in bypass replication of pyrimidine dimers in the cancer-prone hereditary disease xeroderma pigmentosum variant (XPV). An experimental strategy involving in vitro complementation of replication bypass deficient XPV cell-free extracts using purified replication/repair factors or fractionated extracts from HeLa cells will be developed. Using a SV40 based replication system containing a site- and strand-specific UV-induced pyrimidine dimer that was previously utilized in Dr. Vos s laboratory bypass DNA synthesis will be assessed for pyrimidine dimers on the leading DNA strand using four different XPV cell lines (two EBV-transformed lymphoblastoid and two fibroblast lines transformed with replication defective SV40). The second specific aim focuses on analyzing the contribution of error-prone and error-free bypass DNA replication of DNA damage in human cell-free extracts. The contribution of mutagenic translesion synthesis and homologous strand exchange-dependent error-free bypass replication of pyrimidine dimers will be determined. It is hypothesized that if error-free bypass replication of a leading strand T=T occurs, then such DNA synthesis most likely involves sequence information from the opposite undamaged DNA strand. This effort will be conducted in two phases. First, an assay will be developed to distinguish error-prone and error-free DNA synthesis at a site-specific T=T lesion located on the leading DNA strand. Second, the relative contribution of error-prone and error-free replication during bypass will be assessed and used to isolate bypass factors that may be deficient in various cell lines.