Structure of the DNA Repair Enzyme Endonuclease IV and Its DNA Complex (original) (raw)

The first damage-general step of BER, which is conserved from bacteria to humans, is the recognition and cleavage of DNA-abasic sites by an AP endonuclease. There are two characterized conserved AP endonucle-State University of New York-Albany Albany, New York 12222 ase families, and these enzymes cleave the DNA backbone immediately 5Ј of an AP site, generating a 5Ј deoxyribose-phosphate group and a 3Ј deoxyribose-hydroxyl group that primes DNA repair synthesis. The first en-Summary zyme family is typified by Exonuclease III from Escherichia coli (Weiss, 1976; Saporito et al., 1988; Mol Endonuclease IV is the archetype for a conserved apurinic/apyrimidinic (AP) endonuclease family that et al., 1995b) and the homologous APE-1 enzyme in humans (Demple et al., 1991; Gorman et al., 1997), which primes DNA repair synthesis by cleaving the DNA backbone 5 of AP sites. The crystal structures of En-are major AP endonucleases in these organisms. The second conserved AP endonuclease family is typified donuclease IV and its AP-DNA complex at 1.02 and 1.55 Å resolution reveal how an ␣ 8 ␤ 8 TIM barrel fold by E. coli Endonuclease IV (Endo IV) (Saporito and Cunningham, 1988) and includes enzymes thus far identified can bind dsDNA. Enzyme loops intercalate side chains at the abasic site, compress the DNA backbone, bend in several eubacteria and eukarya (Demple et al., 1997; Ramotar, 1997; Haas et al., 1999), including the APN-1 the DNA ‫,؇09ف‬ and promote double-nucleotide flipping to sequester the extrahelical AP site in an enzyme protein from Saccharomyces cerevisiae (Popoff et al., 1990) and Schizosaccharomyces pombe (Ramotar et pocket that excludes undamaged nucleotides. These structures suggest three Zn 2؉ ions directly participate al., 1998) and the CeAPN1 gene from the nematode Caenorhabditis elegans (Masson et al., 1996). In E. coli, in phosphodiester bond cleavage and prompt hypotheses that double-nucleotide flipping and sharp bend-Endo IV expression is induced by superoxide anion generators (Chan and Weiss, 1987; Walkup and Kogoma, ing by AP endonucleases provide exquisite damage specificity while aiding subsequent base excision re-1989), but in S. cerevisiae, APN-1 is the predominant constitutive AP endonuclease. The importance of this pair pathway progression. enzyme for DNA repair in yeast is underscored by genetic experiments that show APN-1 null mutants to be strong mutators driving AT→GC transversions at a rate Introduction ‫-06ف‬fold greater than observed in wild-type cells (Kunz et al., . The purified enzyme specifically cleaves the DNA DNA base damage, and the primary defense against these genotoxic insults is the DNA base excision repair backbone at AP sites and also removes 3Ј DNA-blocking groups such as 3Ј phosphates, 3Ј phosphoglycolates, (BER) pathway (Lindahl, 1993). The first step of BER is initiated by many distinct DNA glycosylases that each and 3Ј ␣,␤-unsaturated aldehydes that arise from oxidarecognize a specific class of damaged DNA nucleotide tive base damage and the activity of combined glycosyand cleave the N-C1Ј glycosidic bond, linking the aberlase/lyase enzymes (Ramotar et al., 1991; Demple and rant base to the deoxyribose sugar (Cunningham, 1997; Harrison, 1994). Endo IV is also the only known repair Krokan et al., 1997). These damage-specific glycosyenzyme that is able to cleave the DNA backbone 5Ј of lases generate as a common product apurinic/apyrimithe oxidative lesion ␣-deoxyadenosine, which arises by dinic (AP or abasic) sites, which are inherently toxic and hydroxyl radical abstraction of a proton from the C1Ј mutagenic Loeb, 1985; Demple et al., 1986; Kingma atom of deoxyribose (Ide et al., 1994). et al., 1995) and thus must be rapidly processed and Our understanding of AP endonuclease recognition removed. In the subsequent damage-general steps of and cleavage of DNA at AP sites lacks key structural single nucleotide BER, an AP endonuclease cleaves the data for either conserved enzyme family bound to DNA. DNA backbone at AP sites, providing a product that is Based on biochemical and mutagenesis results and the crystal structures of Exo III (Mol et al., 1995b) and APE-1 (Gorman et al., 1997) without bound DNA, four hypothe- ‡ To whom correspondence should be addressed (e-mail: jat@ scripps.edu).