Our genetic integrity is assured in part by the precise repair of DNA damage caused by ionizing radiation, chemical mutagens, or the spontaneous hydrolytic decay of DNA bases. If not corrected, these chemical modifications may cause a lethal block of DNA replication or create a mutation because of the ambiguous coding potential of these modified bases. Repair is accomplished by one of several lesion-specific mechanisms including the reversal of the chemical modification, excision of modified bases (base excision repair), or the removal of the DNA strand containing the lesion (nucleotide excision repair). Defects in DNA repair are present in human diseases such as Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, Cockayne syndrome, and Bloom syndrome (Barnes et al, 1993; Kunkel, 1993; Cleaver, 1994), including that these repair processes are a prerequisite to normal human health. Enzymes catalyzing nucleotide excision repair are best characterized in E. coli, in which several DNA N-glycosylases have been identified. These proteins specifically recognize modified bases and cleave the N- glycosylic bond, releasing the damaged base from the DNA backbone. We are pursuing crystallographic and biochemical studies of two purine-specific DNA glycosylases, E. coli DNA glycosylase II (AIkA) and formamidopyrimidine-DNA glycosylase (Fpg protein), which catalyze the removal of alkylated and oxidized purines from DNA. The crystal structure of AIkA is being determined by a combination of multiple isomorphous replacement and multiwavelength anomalous diffraction methods. X-ray data extending to 2.1 Angstroms resolution have been collected from native AIkA crystals, and we have identified several heavy atom derivatives. We are also studying the interaction of methylated purines with the AIkA protein by crystallographic and biochemical methods. These studies are aimed at the identification of the enzyme active site. Crystallization experiments with the Fpg protein are at a more preliminary stage. Crystal structures of AIkA and Fpg proteins and complexes various purine analogs will address substrate recognition and provide the basis for detailed mechanistic studies of these DNA repair enzymes.
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