DNA repair and recombination are fundamental processes underlying cellular responses to environmental and endogenous stresses. Understanding the functions of genes involved in DNA repair and recombination is critical to understanding the molecular basis of mutagenesis and the origins of gene rearrangements, mitotic recombination and carcinogenesis. In Project 4, we will experimentally define some of the relationships among a subset of mammalian DNA repair genes, components of the nucleotide excision repair (NER), double-strand break repair (DSBR), and mismatch repair (MMR) pathways, which are known to function in both repair and recombination. We hypothesize that many of the proteins encoded by these genes function at multiple steps in repair processing of DNA interstrand crosslinks (ICLs) and other types of complex DNA damage in mammalian cells. The specific focus of Project 4 is the function of these genes in recombinational repair. In yeast, the RAD (radiation-sensitive) genes participate in a variety of DNA repair pathways, including NER and DSBR, and also constitute recombinational repair pathways. Studies in yeast have provided clues to how mammalian RAD homologs function in repair and recombination in mammalian cells, but there are also important differences between lower and higher eukarotes in DNA repair and recombination mechanisms; also, repair of complex lesions such as ICLs by mammalian cells is poorly understood. The increased complexity of both repair and recombination in mammalian cells is reflected in the number of paralogs, as well as true orthologs, of many RAD genes. These components form different complexes functioning in repair and recombination, and likely impart both significant and subtle differences to mammalian DNA repair processes compared to yeast. In Project 4, we will construct DNA repairproficient and -deficient CHO cell lines with specifically defective genes (i.e., ERCC1, XPF, RAD52, MSH3, XRCC3 and RAD51C); these cell lines will contain intrachromosomal APRT direct-repeat recombination substrates, and corresponding isogenic cell lines will contain APRT alleles suitable as recombination targets. These specialized cell lines will be used to investigate and compare recombinational repair of ICLs and DSBs, focusing on RAD51-dependent and -independent recombinational repair pathways, and will also be used to investigate genetic interactions through epistasis experiments. Project 4 will generate results addressing the overall hypothesis that repair of complex lesions in mammalian cells involves components of multiple repair pathways acting at multiple steps, including elements of NER, DSBR, and MMR pathways.
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