This proposal focuses on developing an in vitro model to study how DNA replication and repair enzymes act on defined site-directed lesions in DNA. Little is known concerning the repertoire of DNA metabolizing enzymes available in neurons and astrocytes, and how these enzymes respond to DNA damage and vary with age. Two extremely sensitive assays, based on polyacrylamide gel electrophoresis, will be used to study the behavior of DNA synthetic and repair enzymes with three classes of DNA lesions, an abasic (apurinic/apyrimidinic) lesion, alkylated guanine and thymine nucleotide analogues, and 8-oh guanine, a lesion resulting from oxidative damage. The first assay will be used to identify and characterize replication and repair DNA polymerases, using extracts from liver and brian in young and old animals, and in dividing and nondividing cells grown in culture. There is a paucity of data on the ability of the various DNA polymerases to copy and bypass base and sugar modified DNA lesions. The second assay will be used to identify enzymes, such as glycosylases, methyltransferases, endo- and exonucleases, and ligases, involved in lesion repair. We intend to assay crude extracts of brain and liver cells for activities that are induced in response to DNA damage, and to purify and characterize these enzymes. We will determine if animal cells exhibit an induced """"""""error-prone"""""""" response to DNA damage analogous to the SOS response in Escherichia coli and other enterobacteria.
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