GENOMIC DISTRIBUTION AND PHYLOGENY OF DNA REPAIR

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We will determine whether base excision repair of oxidatively damaged DNA bases occurs preferentially in actively transcribed genes. The repair of 5-hydroxymethyluracil (HmUra), which is repaired by HmUra-DNA glycosylase and does not block replication, will be compared to that of thymine glycol and related lesions which are repaired by E. coli endonuclease III and do block replication. HmUra-DNA glycosylase is unique among repair enzymes in being absent from bacteria and yeast. We have proposed that its phylogenetic distribution reflects its major function which is the repair of 5-methylcytosine residues in DNA. In contrast, endonuclease III-like activities are phylogenetically conserved indicating that the lesions repaired by the enzymes are formed in all species. Among these lesions are UV induced pyrimidine hydrates. The hydration of cytosine leads to irreversible deamination to uracil hydrate which, if unrepaired, leads to point mutations. This reaction scheme may also occur for 5-methylcytosine residues leading to irreversible deamination to thymine hydrate. We will study the formation and stability of pyrimidine photohydrates in oligonucleotides of high specific radioactivity using E. coli endonuclease III as a probe. We will use these oligonucleotides as substrates for the purification of mammalian endonuclease III-like activities to a purity sufficient to obtain protein sequence and molecular cloning. The results of these experiments will add to our knowledge of the formation and repair of oxidative and photochemical damage to DNA and possibly elucidate the relationship of such damage to the etiology of actinic and spontaneous cancer.