The objective of this research is to understand how cell cycle-dependent variation in repair activity, gene expression, nuclear matrix association, and replication affect DNA repair and the differential processing of DNA damage in the mammalian genome, Our long-term goal is to define the dynamic relationships between cell cycle-dependent variation in gene expression and DNA repair, and their ultimate biological consequences regarding cell survival and mutagenesis. We will characterize variations in DNA repair capacity and repair gene expression during the Chinese hamster ovary (CHO) cell cycle. This will be accomplished by performing host cell reactivation experiments using a transient expression vector introduced by electroporation into synchronous populations. cell populations for synchrony experiments will be obtained by use of the mitotic cell selection and/or elutriation centrifugation. Also by using cloned mammalian repair gene sequences as probes we will measure the constitutive and inducible levels of transcription of DNA repair genes in synchronous cell population. We will also investigate efficiency or repair of specific genes as a function of growth status and cell cycle phase. and seek correlations with the timing of transcription and replication of these genes, and their temporal association with the nuclear matrix during progression through the cell cycle. Finally, using such methods as the polymerase chain reaction procedure we will examine how the spectrum and strand-specificity of UV-induced mutations at the CHO APRT locus are affected by DNA repair capacity and possible differences in the rates of repair of transcribed and non-transcribed strands. The result of these investigations will enhance our knowledge of the processing of DNA damage in the mammalian cell genome, and will lead to a more detailed description of the molecular basis for the heterogeneity observed in mammalian DNA repair. Since it is generally appreciated that the cytotoxic and mutagenic effects of UV radiation are fundamental components of sunlight-induced human skin cancers, the results of these studies will be directly relevant to a more complete understanding of the mechanisms of sunlight carcinogenesis.
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