The broad objective of this grant is to elucidate the means by which damaged DNA is copied by the cell's DNA replication and repair enzymes, focusing primarily on proteins that are induced in response to DNA damage. Damage-induced DNA repair occurs in both procaryotic and eucaryotic organisms. In Escherichia coli, response to DNA damage is orchestrated by an operon, the """"""""SOS regulon"""""""", that contains at least 25 different proteins under negative control of a repressor protein, LexA, and a multifunctional protein, RecA. In E. coli, and possibly in animal cells, damage-induced DNA repair is aberrant. That is, it exhibits reduced fidelity while enabling replication to continue past normally blocking DNA damage sites. There are to major specific goals of this proposal. The first is to elucidate the biochemical basis for SOS-induced error-prone repair in E. coli. There are numerous types of damage that occur in DNA when cells are exposed to chemicals, drugs or radiation. Damage to DNA can also occur by naturally occurring spontaneous processes. In setting up biochemical systems to study error-prone repair in vitro and in vivo, we have chosen to focus our attention on copying a single site-directed abasic (apurinic/apyrimidinic) DNA lesion, a biologically relevant noncoding lesion that can occur by spontaneous and induced mechanisms. The absence of a coding base in DNA presents a strong block to replication. When replication past an abasic lesion does occur, it generally results in a mutation. We want to quantify the effects of damage-induced repair proteins in E. coli, specifically, DNA polymerases II, RecA, UmuC, UmuD, and the replication polymerase, DNA polymerase III, on nucleotide insertion and excision (proofreading) at the site of the lesion, and bypass of the lesion. We are proposing to use a novel fluorescence assay to measure the dynamics of forming a lesion-dependent protein complex, on a pre-steady-state time scale. The second major goal of this proposal is to determine the structure and function of DNA polymerase II in E. coli. Although this enzyme was discovered more than 20 years ago, a clear role for it has not been established. Pol II is clearly important because it is induced in response to DNA damage. It is also unusual because it shares significant amino acid sequence similarity with the class of eucaryotic alpha-DNA polymerases. We intend to study the enzymatic, genetic, and structural properties of Pol II. We have recently obtained X-ray crystallographic data with Pol II that will be used in conjunction with biochemical and kinetic data to establish a detailed mechanism of action for this enzyme, and to make a detailed comparison of Pol II structure with E. coli Pol I (large fragment), and HIV-1 reverse transcriptase.
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