The overall goal of this application is to understand the in vivo role that DNA alkylation repair pathways play in mammals. The model organism to be employed in these studies is the mouse. This research team has recently cloned and characterized two mammalian DNA alkylation repair genes, namely the 3-methyladenine DNA glycosylase gene (Aag) and O6-methylguanine DNA repair methyltransferase gene (Mgmt). They have used these cloned genes to make targeting constructs for their mutation in pluripotent mouse embryonic stem (ES) cells by targeted homologous recombination. The ES cells were used to produce mice that are heterozygous and homozygous for the mutations in the Aag and Mgmt genes. It is the contention of these investigators that they now have all the tools necessary for investigating the role of these two DNA repair pathways in protecting mice against the toxic, mutagenic, clastogenic, and carcinogenic effects of a variety of DNA alkylating agents. These agents exist in the environment, they are formed in the animal as normal cellular metabolites, and they are commonly used for cancer chemotherapy. Additionally, it is proposed to cross the mice produced here with other strains that are defective in certain other DNA repair pathways, and to assess their relative contributions to protecting the animal. Also, these mice will be crossed with p53 and Atm null cancer-prone mice to determine whether there is any interaction of the p53 and Atm- regulated checkpoints with DNA alkylation damage. Finally, it is proposed to express the human AAG and MGMT cDNAs in these knockout mice to assess what role the human enzymes are capable of playing in animals.
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