PROCESSING OF OXIDATIVE DNA LESIONS--TRANSIENT KINETICS

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date4/1/9811/30/98

Funding

  • National Cancer Institute

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