Specific Recognition of Substrate Analogs by the DNA Mismatch Repair Enzyme MutY

Porello, Silvia L.; Williams, S. D.; Kuhn, Heiko; Michaels, Mark L.; David, Sheila S.

doi:10.1021/ja9602206

Cited by 81 publications

(141 citation statements)

References 47 publications

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“…3). No electrochemical signal for MutY is observed at an electrode modified with the duplex SH-5Ј-AGTACAGTCATCGCG hybridized to a complement incorporating an abasic site opposite the underlined cytosine (64). It has been established that the presence of an intervening abasic site or other DNA base-stacking perturbation attenuates the reduction of intercalators bound to DNA films (50,52,57).…”

Section: Resultsmentioning

confidence: 99%

DNA-mediated charge transport for DNA repair

Boon

Livingston

Chmiel

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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223

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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....

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Section: Resultsmentioning

confidence: 99%

DNA-mediated charge transport for DNA repair

Boon

Livingston

Chmiel

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

223

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“…This was explored in two separate experiments. MutY is known to bind preferentially to a 7-deazaadenine:guanine (ZG) base pair without excising the modified adenine [74]. We observe that addition of MutY to the assembly containing the ZG base pair located 19 base pairs from the electron trap leads to a significantly greater regeneration of the EPR signal when compared to an identical duplex containing a CG base pair at the analogous site.…”

Section: Modified Nitroxyl Radical As An Electron Trap In Dna-mediatementioning

confidence: 88%

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

Boal

Yavin

Barton

2007

Journal of Inorganic Biochemistry

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The [4Fe-4S] cluster is ubiquitous to a class of base excision repair enzymes, in organisms ranging from bacteria to man, and was first considered as a structural element, owing to its redox stability under physiological conditions. When studied bound to DNA, two of these repair proteins (MutY and Endonuclease III from Escherichia coli) display DNA-dependent reversible electron transfer with characteristics typical of high potential iron proteins. These results have inspired a reexamination of the role of the [4Fe-4S] cluster in this class of enzymes. Might the [4Fe-4S] cluster be used as a redox cofactor to search for damaged sites using DNA-mediated charge transport, a process well known to be highly sensitive to lesions and mismatched bases? Described here are experiments demonstrating the utility of DNA-mediated charge transport in characterizing these DNA-binding metalloproteins, as well as efforts to elucidate this new function for DNA as an electronic signaling medium among the proteins.

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“…With duplexes containing G:A and G:Z3 mismatches, the affinity was approximately three-orders of magnitude smaller and only slightly higher (~5-fold) than that observed with a duplex lacking a mismatch (K d = 150 ± 60 nM). 39 This suggests that the better predictor of high levels of repair is efficient binding of MutY to the mismatch. We suggest that affinity of MutY for a given mismatch may be reporting on the ability of MutY to efficiently locate the mismatch.…”

Section: Discussionmentioning

confidence: 99%

“…17 For E37S MutY, binding titrations were first performed to determine the amount of "active" enzyme. 18 Glycosylase activity assays and dissociation constants (K d ) were measured as described previously 17,31,39 . Buffer conditions for the glycosylase assays were 20 mM Tris-HCl pH 7.6, 10 mM EDTA, 0.1 mg/mL BSA, and 30 mM or 150 mM NaCl.…”

Section: Muty Purification Glycosylase and Binding Assaysmentioning

confidence: 99%

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

et al. 2007

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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....

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Specific Recognition of Substrate Analogs by the DNA Mismatch Repair Enzyme MutY

Cited by 81 publications

References 47 publications

DNA-mediated charge transport for DNA repair

DNA-mediated charge transport for DNA repair

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

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