SITE SPECIFIC DNA DAMAGE AND GENOMIC INSTABILITY IN MAMM

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DNA damage by agents present in the environment often leads directly, or indirectly, to DNA strand breaks. The biological consequences of some environmental insults, such as ionizing radiation, seem to derive primarily from DNA strand breaks introduced into chromosomes. Other environmental agents, such as ultraviolet light and agents that introduce base modifications, may indirectly lead to DNA strand breaks and gaps as intermediates in the DNA repair process. Double-strand breaks in chromosomes are potentially lethal to the cell. If they are not repaired, breaks can lead to the loss of chromosome segments or whole chromosomes; if misrepaired, they can lead to the rearrangement of genetic information. The processes responsible for the repair of this damage in mammalian cells by homologous and nonhomologous (illegitimate) recombination are poorly defined and difficult to study because there are few ways to introduce site-specific DNA damage in a controlled fashion; and there are few well-characterized genomic sites that are useful for studying site-specific DNA damage. The proposed research uses a well- established mammalian cell genetic system, the hamster adenine phosphoribosyltransferase (APRT) gene, that has been modified to study homologous and illegitimate recombination. The specific aims of the research are: 1) To characterize the physical processing of site-specific DNA strand breaks in mammalian cells. Site-specific double- and single-strand breaks will be introduced by introducing recognition sites for proteins that introduce strand breaks into the chromosomal APRT gene and expression of those proteins in the modified cell lines. Intermediates in the processing of DNA strand breaks will be detected using PCR and Southern analysis. 2) To better understand the genetic consequences of DNA strand breaks. The influence of DNA strand breaks on mutagenesis in surrounding sequences will be determined by selection for mutations in selectable markers close to APRT and characterization of the mutations by DNA sequencing. 3) To investigate the role of the structure-specific nucleases, ERCC1/XPF, XPG, and FEN-1 on processing DNA structures and genomic instability.