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 bacterial second messenger c-di-GMP is used in many species to control essential processes that allow the organism to adapt to its environment. The c-di-GMP riboswitch (GEMM) is an important downstream target in this signaling pathway and alters gene expression in response to changing concentrations of c-di-GMP. The riboswitch selectively recognizes its second messenger ligand primarily through contacts with two critical nucleotides. However, these two nucleotides are not the most highly conserved residues within the riboswitch sequence. Instead, nucleotides that stack with c-di-GMP and that form tertiary RNA contacts are the most invariant. Biochemical and structural evidence reveals that the most common natural variants are able to make alternative pairing interactions with both guanine bases of the ligand. Additionally, a high resolution (2.3 Å) crystal structure of the native complex reveals that a single metal coordinates the c-di-GMP backbone. Evidence is also provided that after transcription of the first nucleotide on the 3′-side of the P1 helix, which is predicted to be the molecular switch, the aptamer is functional for ligand binding. Although large energetic effects occur when several residues in the RNA are altered, mutations at the most conserved positions, rather than at positions that base pair with c-di-GMP, have the most detrimental effects on binding. Many mutants retain sufficient c-di-GMP affinity for the RNA to remain biologically relevant, which suggests that this motif is quite resilient to mutation.

DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash͞ quench technique, is found to promote oxidation of the 1؉ clusters. In ruthenium-tethered DNA assemblies, oxidative damage to the 5-G of a 5-GG-3 doublet is generated from a distance but this irreversible damage is inhibited by MutY and instead EPR experiments reveal cluster oxidation. With rutheniumtethered assemblies containing duplex versus single-stranded regions, MutY oxidation is found to be mediated by the DNA duplex, with guanine radical as an intermediate oxidant; guanine radical formation facilitates MutY oxidation. A model is proposed for the redox activation of DNA repair proteins through DNA CT, with guanine radicals, the first product under oxidative stress, in oxidizing the DNA-bound repair proteins, providing the signal to stimulate DNA repair.electron transfer ͉ iron-sulfur cluster ͉ oxidative DNA damage D NA-mediated charge transport (CT) from a distance to generate oxidative damage was first demonstrated in an assembly containing a tethered metallointercalator (1). In this assembly, photoinduced oxidative damage of the 5Ј-G of 5Ј-GG-3Ј sites was observed; this damage pattern has since become the hallmark of DNA CT chemistry, and long-range oxidative damage has been confirmed by using a variety of pendant oxidants (2-6). Long-range oxidative DNA damage has been demonstrated over a distance of at least 200 Å (7, 8). Indeed, DNA either packaged in nucleosome core particles (9) or inside the cell nucleus (10) has been found to be susceptible to long-range oxidative damage. Chemically well defined assemblies, consisting of DNA duplexes with covalently bound oxidants, have been particularly useful in establishing the sensitivity of DNA CT to base-stacking perturbation (11-16). Recently, analogous studies probing long-range reductive chemistry on DNA has been probed both in solution (17-20) and on DNAmodified surfaces (14,15,21). As with oxidation chemistry, these reactions show only small variations in rate with distance but are remarkably sensitive to perturbations in the intervening base pair stack. Mechanistic descriptions for DNA CT focused first on a mixture of hopping and tunneling. A phonon-assisted polaron model has also been put forth (22). Studies as a function of temperature have shown the CT process to be gated by base pair dynamics; in fact, base pair motions are required for CT (23, 24).We have therefore described DNA CT in the context of transport among delocalized DNA domains formed and dissolved based on sequence-dependent DNA dynamics.Given the exquisite sensitivity of DNA CT to DNA lesions and mismatches, we have recently explored a possible role for DNA CT in repair. We demonstrated that redox activity required DNA binding for MutY (25), a base excision repair (BER) enzyme fr...

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Escherchia coli MutY plays an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG) in DNA by excising adenines from OG:A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG:A substrate on the kinetics of base removal, mismatch affinity and repair to G:C in an Escherchia coli-based assay. Surprisingly, adenine modification was tolerated in the cellular assay, while modification of OG results in minimal cellular repair. High affinity for the mismatch and efficient base removal require the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature for MutY to locate OG:A mismatches and select the appropriate adenines for excision to initiate repair in vivo prior to replication.The mismatch repair (MMR) pathway in E. coli relies on methylation at a GATC sequence to direct the repair machinery to remove the mismatched base on the newly synthesized strand. 1 However, almost two decades ago, an activity was detected in E. coli cell extracts that restored G:A mismatches to G:C matches and was insensitive to the methylation state of the template strand. [2][3][4] Concurrently, a mutator locus in E. coli (mutY) was identified that generated G:C → T:A transversion mutations. 5 It was later determined that the mutY gene product is an adenine glycosylase capable of removing adenines from G:A mismatches as the first step in base excision repair (BER). [6][7][8][9][10] The subsequent activity of downstream BER pathway enzymes, e.g. the AP endonuclease, deoxyribophosphate lyase, polymerase and ligase, restores the G:C base pair in a methylation-independent fashion. 11 MutY was also found to participate in the prevention of mutations caused by 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG, 1) by removal of adenine from OG:A mismatches. 10,12,13,14 Polymerase misinsertion of dAMP opposite OG creates OG:A mismatches that can lead to formation of T:A base pairs upon a second round of replication. 12 Recently, MutY has been in the spotlight due to the correlation between inherited biallelic mutations in the gene encoding the human homologue of MutY (MUTYH) and colorectal cancer. 10,15,16 *Author to whom correspondence should be addressed. Email: david@chem.ucdavis.edu,. # These authors contributed equally to this manuscript. We have previously examined the features of mismatched substrates that are required for efficient lesion recognition and adenine excision by MutY using pre-steady state and singleturnover kinetics. [17][18][19] Pre-steady state experiments revealed that MutY has a high affinity for the product such that release of the DNA product is rate-limiting. 17,19,20 Moreover, the identity of the base opposite A greatly affects both the rate of product release as well as the intrinsic rate of adenine removal determined under single-turnover conditions....

MUTYH-associated polyposis (MAP) is the only inherited colorectal cancer syndrome that is associated with inherited biallelic mutations in a base excision repair gene. The MUTYH glycosylase plays an important role in preventing mutations associated with 8-oxoguanine (OG) by removing adenine residues that have been misincorporated opposite OG. MAP-associated mutations are present throughout MUTYH, with a large number coding for missense variations. To date the available information on the functional properties of MUTYH variants is conflicting. In this study, a kinetic analysis of the adenine glycosylase activity of MUTYH and several variants was undertaken using a correction for active fraction to control for differences due to overexpression and purification. Using these methods, the rate constants for steps involved in the adenine removal process were determined for the MAP variants Y165C, G382D, P391L and Q324R MUTYH. Under single-turnover conditions, the rate of adenine removal for these four variants was found to be 30-40% of WT MUTYH. In addition, the ability of MUTYH and the variants to suppress mutations and complement for the absence of MutY in E. coli was assessed using rifampicin resistance assays. The presence of WT and Q324R MUTYH resulted in complete suppression of the mutation frequency, while G382D MUTYH showed reduced ability to suppress the mutation frequency. In contrast, the mutation frequency observed upon expression of P391L and Y165C MUTYH were similar to the controls, suggesting no activity toward preventing DNA mutations. Notably, though all variations studied herein resulted in similar reductions in adenine glycosylase activity, the effects in the bacterial complementation are quite different. This suggests that the consequences of a specific amino acid variation on overall repair in a cellular context may be magnified.

The oxidation product of 2'-deoxyguanosine, 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG), produces G:C to T:A transversion mutations. The Escherichia coli base excision repair glycosylase MutY plays an important role in preventing OG-associated mutations by removing adenines misincorporated opposite OG lesions during DNA replication. Recently, biallelic mutations in the human MutY homologue (hMYH) have been correlated with the development of colorectal cancer. The two most common mutations correspond to two single amino acid substitutions in the hMYH protein: Y165C and G382D [Al-Tassan, N., et al. (2002) Nat. Genet. 30, 227-232]. Previously, our laboratory analyzed the adenine glycosylase activity of the homologous variant E. coli MutY enzymes, Y82C and G253D [Chmiel, N. H., et al. (2003) J. Mol. Biol. 327, 431-443]. This work demonstrated that both variants have a reduced adenine glycosylase activity and affinity for substrate analogues compared to wild-type MutY. Recent structural work on Bacillus stearothermophilus MutY bound to an OG:A mismatch-containing duplex indicates that both residues aid in recognition of OG [Fromme, J. C., et al. (2004) Nature 427, 652-656]. To determine the extent with which Tyr 82 and Gly 253 contribute to catalysis of adenine removal by E. coli MutY, we made a series of additional modifications in these residues, namely, Y82F, Y82L, and G253A. When the substrate analogue 2'-deoxy-2'-fluoroadenosine (FA) in duplex paired with G or OG is used, both Y82F and G253A showed reduced binding affinity, and G253A was unable to discriminate between OG and G when paired with FA. Additionally, compromised glycosylase activity of Y82F, Y82C, and G253A MutY was observed using the nonoptimal G:A substrate, or at low reaction temperatures. Interestingly, adenine removal from an OG:A-containing DNA substrate by Y82C MutY was also shown to be extremely sensitive to the NaCl concentration. The most surprising result was the remarkably similar activity of Y82L MutY to the WT enzyme under all conditions examined, indicating that a leucine residue may effectively replace tyrosine for intercalation at the OG:A mismatch. The results contained herein provide further insight regarding the intricate roles of Tyr 82 and Gly 253 in the OG recognition and adenine excision functions of MutY.