Nucleotide excision repair is the major cellular defense mechanism against internal and external DNA damaging agents; by removing modified nucleotides from DNA it prevents mutation, cancer and cellular death. The goal of our research is to understand the molecular mechanisms of this phenomenon in the biological world by investigating it in organisms ranging from a Mycoplasma, which has the smallest genome of any free living species, to E. coli, and humans. (I). M. GENITALIUM. We will clone the three genes of the subunits of the excision nuclease, overproduce, purify and characterize the subunits, and reconstitute the enzyme. We will determine the mode of damage removal by the reconstituted enzyme. (II). E. COLI. a) Structure. The E. coli (A)BC excinuclease is an ATP -dependent nuclease which removes damaged nucleotides from DNA by hydrolyzing the 8th phosphodiester bond 5' and the 4th phosphodiester bond 3' to the lesion. The enzyme is made up of UvrA, UvrB, and UvrC subunits. The domains and residues of all three, involved in subunit-subunit interactions and in enzymesubstrate interaction will be identified by genetic engineering and physical methods. b) The mechanism of damage recognition will be investigated by foot-printing, fluorescence quenching, site-specific mutagenesis, psoralen crosslinking, and high-resolution electron microscopy. c) Catalysis. The subunit(s) directly responsible for hydrolyzing the two phosphodiester bonds and the amino acids involved in forming two catalytic triads will be identified by chemical modification and site specific mutagenesis. d) Turnover. Catalytic turnover of the subunits by Pol I and helicase II, and postincision gap filling will be analyzed by kinetic and equilibrium methods, DNA-protein crosslinking, and footprinting techniques. e) Gene and strand specific repair will be reconstituted in vitro and the 'transcription-repair coupling factor' will be purified and the coupling mechanism investigated. (III). HUMAN excision nuclease. a) The system. We have developed an in vitro nucleotide excision repair system. The relation of this activity to XP (xeroderma pigmentosum) gene products (required for repair in vivo) will be established. b) Substrate. To characterize the enzyme(s), uniquely modified substrate plasmids will be prepared. c) Assays that measure the binding, incision, excision, and resynthesis steps of repair will be developed and the exact mode of incision and adduct removal will be determined. d) The subunits of the excision nuclease(s) will be purified from HeLa cells by standard and FPLC chromatography. DNA binding, ATPase, and nicking activities will be ascribed to individual proteins. e) The XP genes of complementation groups XP-A, -B, -C, -D, and -E will be cloned and the gene products will be overproduced, purified and characterized.
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