Inherited defects of base excision repair have not been associated with any human genetic disorder, although mutations of the genes mutM and mutY, which function in Escherichia coli base excision repair, lead to increased transversions of G:C to T:A. We have studied family N, which is affected with multiple colorectal adenomas and carcinoma but lacks an inherited mutation of the adenomatous polyposis coli gene (APC) that is associated with familial adenomatous polyposis. Here we show that 11 tumors from 3 affected siblings contain 18 somatic inactivating mutations of APC and that 15 of these mutations are G:C-->A transversions--a significantly greater proportion than is found in sporadic tumors or in tumors associated with familial adenomatous polyposis. Analysis of the human homolog of mutY, MYH, showed that the siblings were compound heterozygotes for the nonconservative missense variants Tyr165Cys and Gly382Asp. These mutations affect residues that are conserved in mutY of E. coli (Tyr82 and Gly253). Tyrosine 82 is located in the pseudo-helix-hairpin-helix (HhH) motif and is predicted to function in mismatch specificity. Assays of adenine glycosylase activity of the Tyr82Cys and Gly253Asp mutant proteins with 8-oxoG:A and G:A substrates show that their activity is reduced significantly. Our findings link the inherited variants in MYH to the pattern of somatic APC mutation in family N and implicate defective base excision repair in predisposition to tumors in humans.

MutY, like many DNA base excision repair enzymes, contains a [4Fe4S] 2؉ cluster of undetermined function. Electrochemical studies of MutY bound to a DNA-modified gold electrode demonstrate that the [4Fe4S] cluster of MutY can be accessed in a DNA-mediated redox reaction. Although not detectable without DNA, the redox potential of DNA-bound MutY is Ϸ275 mV versus NHE, which is characteristic of HiPiP iron proteins. Binding to DNA is thus associated with a change in [4Fe4S] 3؉/2؉ potential, activating the cluster toward oxidation. Given that DNA charge transport chemistry is exquisitely sensitive to perturbations in base pair structure, such as mismatches, we propose that this redox process of MutY bound to DNA exploits DNA charge transport and provides a DNA signaling mechanism to scan for mismatches and lesions in vivo.D NA repair proteins that contain a FeS redox cofactor are ubiquitous (1-7), yet a role for these factors has been lacking. Two examples, highly homologous (8), are MutY (1) and endonuclease III (Endo III) (2, 9), base excision repair enzymes from Escherichia coli (10). MutY, containing 350 residues, acts as a glycosylase to remove adenine from G:A (11-13) and 7,8-dihydro-8-oxo-2-deoxyguanonsine:A mismatches (14-21); Endo III removes pyrimidines damaged by ring saturation, contraction, or fragmentation (22-29). Although MutY and Endo III have dramatically different substrate recognition features, they both contain a [4Fe4S] 2ϩ cluster (1, 2, 9) within a Cys-X 6 -Cys-X 2 -Cys-X 5 -Cys loop located near the protein C terminus (1, 9, 30). Based on sequence alignment, a loop defined by the first two ligating cysteines, called the FCL, is proposed to be a common element of DNA repair proteins (30), present throughout phylogeny (31-37). The function, if any, for these clusters remains undetermined, although the FCL has been proposed as a structural element, aiding in DNA binding (9,30,38). Interestingly, however, MutY is capable of folding without the cluster; the [4Fe4S] 2ϩ cluster adds no stability to the enzyme, but it is critical for substrate binding and catalysis (39). The solvent-accessible [4Fe4S] 2ϩ cluster of Endo III undergoes decomposition with ferricyanide and is resistant to reduction, with an estimated midpoint potential of ϽϪ600 mV for the [4Fe4S] 2ϩ/1ϩ couple (2, 38).Here, we consider whether the [4Fe4S] 2ϩ cluster in MutY can function in DNA-mediated charge transport (CT). Many laboratories have probed DNA-mediated CT chemistry (40-42). Using biochemical, spectroscopic, and electrochemical methods, we have shown that CT through DNA can proceed over long molecular distances in a reaction that is remarkably sensitive to intervening dynamical base pair structure (43-50). DNAmediated CT has been shown to yield oxidative DNA damage from a distance within nucleosome core particles (47) and HeLa cell nuclei (51). DNA binding proteins and peptides have also been shown to modulate and participate in long-range CT chemistry (48, 49), raising the question of the physiological relevance of DNA CT....

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7, 8-dihydro-8-oxo-2'-deoxyguanosine:2'-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1-Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.

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