Adenine Release Is Fast in MutY-catalyzed Hydrolysis of G:A and 8-Oxo-G:A DNA Mismatches

McCann, Joe A.B.; Berti, Paul J.

doi:10.1074/jbc.m212474200

Cited by 33 publications

(38 citation statements)

References 28 publications

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“…17 The rate constant k 3 measured for the reaction of MutY with the OG:Z3 substrate was essentially identical to that measured with the OG:A substrate under all conditions. This is not unexpected since k 3 is dominated by release of MutY from the DNA product, 20 which is the same for both substrates after base excision and release.…”

Section: Glycosylase Activity Of Muty With Modified Substratesmentioning

confidence: 84%

“…[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. 17 Product release is likely enhanced in vivo by interactions with other BER enzymes.…”

mentioning

confidence: 99%

“…This biphasic profile is due to slow release of MutY from the DNA product. 17,20 Based on this behavior the same minimal kinetic scheme and approach to determine the relevant rate constants as we have described previously was used (Fig. 2c).…”

mentioning

confidence: 99%

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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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Section: Glycosylase Activity Of Muty With Modified Substratesmentioning

confidence: 84%

mentioning

confidence: 99%

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

et al. 2007

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“…[36][37][38] For hTDG, the resulting product inhibition is so potent that it exhibits almost no turnover in vitro. We therefore used single turnover kinetics and saturating enzyme conditions to obtain rate constants (k max ) that were not impacted by product release or the bimolecular association of enzyme and substrate, and thus correspond to the maximal catalytic activity of hTDG ( Figure 3).…”

Section: Single Turnover Kineticsmentioning

confidence: 99%

“…Release of the base (B) likely precedes very slow release of AP DNA (apD). 36,38 The k max values report on the reaction steps from the initial hTDG·DNA collision complex to the ternary product complex. Electrostatic potential maps of the 5-substituted uracil and cytosine bases used in this work.…”

Section: Supplementary Materialsmentioning

confidence: 99%

Specificity of Human Thymine DNA Glycosylase Depends on N-Glycosidic Bond Stability

et al. 2006

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Initiating the DNA base excision repair pathway, DNA glycosylases find and hydrolytically excise damaged bases from DNA. While some DNA glycosylases exhibit narrow specificity, others remove multiple forms of damage. Human thymine DNA glycosylase (hTDG) cleaves thymine from mutagenic G·T mispairs and recognizes many additional lesions, and has a strong preference for nucleobases paired with guanine rather than adenine. Yet, hTDG avoids cytosine, despite the millionfold excess of normal G·C pairs over G·T mispairs. The mechanism of this remarkable and essential specificity has remained obscure. Here, we examine the possibility that hTDG specificity depends on the stability of the scissile base-sugar bond by determining the maximal activity (k max ) against a series of nucleobases with varying leaving group ability. We find that hTDG removes 5-fluorouracil 78-fold faster than uracil and 5-chlorouracil 572-fold faster than thymine, differences that can be attributed predominantly to leaving group ability. Moreover, hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5-hydroxycytosine, indicating that cytosine has access to the active site. A plot of log(k max ) versus leaving group pK a reveals a Brønsted-type linear free energy relationship with a large negative slope of β lg = −1.6 ± 0.2, consistent with a highly dissociative reaction mechanism. Further, we find that the hydrophobic active site of hTDG contributes to its specificity by enhancing the inherent differences in substrate reactivity. Thus, hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site.

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Measurement of translesion synthesis by fluorescent capillary electrophoresis: 7,8-Dihydro-8-oxodeoxyguanosine bypass modulation by natural products

Nachtergael

Charles

Spanoghe

et al. 2013

Analytical Biochemistry

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Adenine Release Is Fast in MutY-catalyzed Hydrolysis of G:A and 8-Oxo-G:A DNA Mismatches

Cited by 33 publications

References 28 publications

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

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

Specificity of Human Thymine DNA Glycosylase Depends on N-Glycosidic Bond Stability

Measurement of translesion synthesis by fluorescent capillary electrophoresis: 7,8-Dihydro-8-oxodeoxyguanosine bypass modulation by natural products

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