PROCESSING OF OXIDATIVE DNA LESIONS--TRANSIENT KINETICS

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DESCRIPTION: The integrity of DNA is continuously threatened by a wide spectru of damaging agents. One of the greatest challenges is oxidation since it is caused by agents ranging from ionizing radiation to normal cellular metabolism and it has been implicated in mutagenesis, carcinogenesis and aging. Whether o not oxidative DNA lesions have permanent biological consequences depends on th interactions between them and the different cellular components responsible fo maintaining the integrity of DNA. These include base excision repair glycosylases and DNA polymerases. DNA N-glycosylases are faced with identifyin a small number of lesions with, often, high structural similarity to their cognate bases in a vast excess of normal nucleotides. This is further complicated by the large, overlapping substrate spectra observed for N-glycosylases, making the process of lesion recognition very interesting. Definition of the transient-state kinetic parameters of the N-glycosylase and AP lyase reactions as well as the influence of lesion structure and sequence context on repair efficiency should contribute significantly to developing a comprehensive mechanistic model for the action of base excision repair enzymes on oxidized DNA. Perhaps the most important determinant of the biologically harmful potential of any lesion is interactions with DNA polymerases since the outcome is often permanent. Much work has been done both in vitro and in vivo to characterize these interactions. We propose to use transient-state kinetics to delineate the mechanism of translesion DNA synthesis and extend the comprehensive model developed for synthesis on natural templates to include oxidative lesion-containing templates. The influence of lesion structure, sequence context and proprieties of the DNA polymerase such as proofreading an processivity on lesion bypass will be addressed using a reconstituted phase T4 DNA polymerase holoenzyme on templates containing one of several oxidative lesions.