A Dimeric Mechanism for Contextual Target Recognition by MutY Glycosylase

Wong, Isaac; Bernards, Andrew S.; Miller, Jamie K.; Wirz, J

doi:10.1074/jbc.m209802200

Cited by 18 publications

(14 citation statements)

References 58 publications

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“…The MutY molecule that does not bind to the mismatched site dissociates from the dimer, and the active MutY molecule remains bound to the DNA for catalysis. Our model is consistent with the findings of Wong et al (52) that MutY assembles into a dimer upon binding DNA and that the dimer is the functionally active form. However, our findings indicate that a MutY dimer is present only transiently while MutY searches for a mismatched site.…”

Section: Discussionsupporting

confidence: 82%

“…Similarly, the Y-DNA1 and Y-DNA2 complexes with the 20-bp DNA fragment are likely PD and P 2 D complexes. Wong et al (52) have reported that MutY assembles into a dimer upon binding 21-mer DNA and that the dimer is in the functionally active form. In their study, the MutY⅐DNA complex resolved on native gel with 50 mM Tris borate (pH 8.3) and 1 mM EDTA was assigned as a dimer⅐DNA complex (P 2 D).…”

Section: Discussionmentioning

confidence: 99%

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An Escherichia coli MutY Mutant without the Six-helix Barrel Domain Is a Dimer in Solution and Assembles Cooperatively into Multisubunit Complexes with DNA

Lee¹,

Bai²,

Houle³

et al. 2004

Journal of Biological Chemistry

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Escherichia coli MutY is an adenine and weak guanine DNA glycosylase involved in reducing the mutagenic effects of 7,8-dihydro-8-oxoguanine (GO). MutY contains three structural domains: an iron-sulfur module, a six-helix barrel module with the helix-hairpinhelix motif, and a C-terminal domain. Here, we demonstrate that the mutant MutY(⌬26 -134), which lacks the six-helix barrel domain, cannot complement the mutator phenotype of a mutY mutant in vivo. However, the mutant can still bind DNA and has weak catalytic activity at high enzyme concentrations. The mutant is a dimer in solution and assembled into two and multiple (up to five) complexes with 20-and 44-bp DNA fragments, respectively, in a concentration-dependent manner. Higher order complexes with DNA substrates containing A/GO mismatches were formed at lower protein concentrations than with the A/G mismatch and homoduplex DNA. Measurement of equilibrium binding using fluorescence anisotropy showed that the mutant protein retains some specificity for A/GO-containing DNA substrates and that the binding event is highly cooperative. This is consistent with the MutY structure determined, which indicates that GO specificity is contributed by both the six-helix barrel and C-terminal domains. The nonspecific binding of MutY(⌬26 -134) to DNA suggests a model in which the specific binding of mismatched DNA by MutY involves sequential interactions, in which one MutY molecule scans the DNA and enhances binding of another MutY molecule to the A/GO mismatch.

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

An Escherichia coli MutY Mutant without the Six-helix Barrel Domain Is a Dimer in Solution and Assembles Cooperatively into Multisubunit Complexes with DNA

Lee¹,

Bai²,

Houle³

et al. 2004

Journal of Biological Chemistry

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“…A very recent all in vitro study with E. coli adenine DNA glycosylase (MutY) showed a dimeric mechanism for contextual target (substrate DNA) recognition. Unlike their pre-steady state enzyme kinetics, suggesting that the MutY utilizes the allosterically regulated dimerization event for target binding prior to initiating the catalytic steps, our results with hNTH1 demonstrated that the dimerization being independent of target binding regulates the turnover of the enzyme in an allosteric manner at the product release step (44). Various other proteins also have been shown to stimulate the activity of hNTH1.…”

Section: Discussionmentioning

confidence: 57%

In Vitro and in Vivo Dimerization of Human Endonuclease III Stimulates Its Activity

Liu

Choudhury

Roy

2003

Journal of Biological Chemistry

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Human endonuclease III (hNTH1), a DNA glycosylase with associated abasic lyase activity, repairs various mutagenic and toxic-oxidized DNA lesions, including thymine glycol. We demonstrate for the first time that the full-length hNTH1 positively cooperates in product formation as a function of enzyme concentration. The protein concentrations that caused cooperativity in turnover also exhibited dimerization, independent of DNA binding. Earlier we had found that the hNTH1 consists of two domains: a well conserved catalytic domain, and an inhibitory N-terminal tail. The N-terminal truncated proteins neither undergo dimerization, nor do they show cooperativity in turnover, indicating that the homodimerization of hNTH1 is specific and requires the N-terminal tail. Further kinetic analysis at transition states reveals that this homodimerization stimulates an 11-fold increase in the rate of release of the final product, an AP-site with a 3 -nick, and that it does not affect other intermediate reaction rates, including those of DNA N-glycosylase or AP lyase activities that are modulated by previously reported interacting proteins, YB-1, APE1, and XPG. Thus, the site of modulating action of the dimer on the hNTH1 reaction steps is unique. Moreover, the high intranuclear (2.3 M) and cytosolic (0.65 M) concentrations of hNTH1 determined here support the possibility of in vivo dimerization; indeed, in vivo protein cross-linking showed the presence of the dimer in the nucleus of HeLa cells. Therefore, it is likely that the dimerization of hNTH1 involving the N-terminal tail masks the inhibitory effect of this tail and plays a critical role in its catalytic turnover in the cell.Reactive oxygen species are generated endogenously in cells as byproducts of oxidative phosphorylation, and enzymatically during inflammatory responses and detoxification reactions. Reactive oxygen species are also generated exogenously because of metal toxicity, ionizing radiation, or in the influence of other environmental agents; reactive oxygen species are by far the most important genotoxic agents that cause various mutagenic, carcinogenic, and cytotoxic lesions in DNA (1). Oxidative DNA damage includes single-and double-strand breaks and a myriad of base lesions. The oxidized base lesions, known to date, are removed from DNA by the base excision repair (BER) 1 pathway. This process is initiated by bifunctional DNA glycosylases that not only catalyze removal of the base lesion but also cause strand cleavage at the resulting apurinic/apyrimidinic (AP) site via ␤-elimination by their associated AP lyase activity (2-4). Among such oxidatively damaged bases, a series of structurally diverse toxic or mutagenic oxidized pyrimidines are repaired by endonuclease III (NTH), a glycosylase/AP lyase present in all species from bacteria through man. Subsequent repair steps include removal of the resulting 3Ј ␣,␤-unsaturated aldehyde by the phosphodiesterase activity of AP-endonuclease (APE), filling of the resulting DNA gap by a DNA polymerase, and, finally, se...

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“…The electrochemistry data indicate that such a DNA-mediated process is possible. Moreover, association of two MutY equivalents on the DNA template has been proposed based on kinetic experiments (79). In the reduced form, the DNA affinity of MutY should be diminished, facilitating dissociation of the protein from its DNA site.…”

Section: Redox Properties Of Muty and Hipip Iron Proteinsmentioning

confidence: 99%

DNA-mediated charge transport for DNA repair

Boon

Livingston

Chmiel

et al. 2003

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

221

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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A Dimeric Mechanism for Contextual Target Recognition by MutY Glycosylase

Cited by 18 publications

References 58 publications

An Escherichia coli MutY Mutant without the Six-helix Barrel Domain Is a Dimer in Solution and Assembles Cooperatively into Multisubunit Complexes with DNA

An Escherichia coli MutY Mutant without the Six-helix Barrel Domain Is a Dimer in Solution and Assembles Cooperatively into Multisubunit Complexes with DNA

In Vitro and in Vivo Dimerization of Human Endonuclease III Stimulates Its Activity

DNA-mediated charge transport for DNA repair

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