Toward a Detailed Understanding of Base Excision Repair Enzymes:  Transition State and Mechanistic Analyses of <i>N</i>-Glycoside Hydrolysis and <i>N-</i>Glycoside Transfer

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

doi:10.1021/cr040461t

Cited by 238 publications

(413 citation statements)

References 507 publications

(1,845 reference statements)

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“…We already cloned and expressed the gene for an AP endonuclease from P. furiosus. 3 We are presently trying to analyze the interaction between PfuAP and PfuPCNA as well as that between PfuUDG and PfuAP.…”

Section: Discussionmentioning

confidence: 99%

Physical and Functional Interactions between Uracil-DNA Glycosylase and Proliferating Cell Nuclear Antigen from the Euryarchaeon Pyrococcus furiosus

Kiyonari

Uchimura

Shirai

et al. 2008

Journal of Biological Chemistry

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Uracil-DNA glycosylase (UDG) is an important repair enzyme in all organisms to remove uracil bases from DNA. Recent biochemical studies have revealed that human nuclear UDG (UNG2) forms a multiprotein complex in replication foci and initiates the base excision repair pathway by interacting with proliferating cell nuclear antigen (PCNA). Here, we show the physical and functional interactions between UDG and PCNA from the hyperthermophilic euryarchaeon, Pyrococcus furiosus. The physical interaction between the two proteins was identified by a surface plasmon resonance analysis. Furthermore, the uracil glycosylase activity of P. furiosus UDG is stimulated by P. furiosus PCNA (PfuPCNA) in vitro. This stimulatory effect was observed only when wild type PfuPCNA, but not a monomeric PCNA mutant, was present in the reaction. Mutational analyses revealed that our predicted PCNA-binding region (AKTLF) in P. furiosus UDG is actually important for the interaction with PfuPCNA. This is the first report describing the functional interaction between archaeal UDG and PCNA.

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Section: Discussionmentioning

confidence: 99%

Physical and Functional Interactions between Uracil-DNA Glycosylase and Proliferating Cell Nuclear Antigen from the Euryarchaeon Pyrococcus furiosus

Kiyonari

Uchimura

Shirai

et al. 2008

Journal of Biological Chemistry

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“…Thus, our findings suggest the hTDG-catalyzed reaction follows a highly dissociative or perhaps a stepwise mechanism, consistent with previous work showing that nearly all enzymatic and non-enzymatic O-and N-glycoside hydrolyses proceed through a highly dissociative A N D N (S N 2) or a stepwise D N *A N (S N 1) mechanism. 24 The Brønsted-type LFER shows that hTDG activity (k max ) is highly correlated with the N1 acidity of the target base in aqueous solution. However, structural studies indicate that the hTDG active site is hydrophobic, 20,21 and nucleobase acidity varies with the polarity of the environment.…”

Section: Linear Free Energy Relationships For N-glycosidic Bond Hydromentioning

confidence: 99%

“…Thus, our findings are consistent with previous work showing that nearly all enzymatic and non-enzymatic O-and N-glycoside hydrolyses follow a highly dissociative A N D N (S N 2) or stepwise D N *A N (S N 1) mechanism. 24 It is of interest to consider how hTDG could stabilize a highly dissociative transition state. The precedent for enzymatic N-glycosidic bond hydrolysis in DNA is the reaction catalyzed by uracil DNA glycosylase (UDG), which proceeds through a stepwise mechanism involving an oxacarbenium cation-uracil anion intermediate.…”

Section: Implications Of the Brønsted-type Lfer For The Mechanism Of mentioning

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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“…Many previous studies show that hydrolysis of the N-glycosylic bond follows a highly dissociative or stepwise mechanism for both enzymatic and noncatalyzed reactions (45). The reactions catalyzed by UDG and MutY (from E. coli) are stepwise, where rupture of the N-glycosylic bond precedes the addition of the nucleophile (water) (46 -48).…”

Section: Asn 140 Is Essential For the Chemicalmentioning

confidence: 99%

Role of Two Strictly Conserved Residues in Nucleotide Flipping and N-Glycosylic Bond Cleavage by Human Thymine DNA Glycosylase

Maiti

Morgan

Drohat

2009

Journal of Biological Chemistry

119

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Thymine DNA glycosylase (TDG) promotes genomic integrity by excising thymine from mutagenic G⅐T mismatches arising by deamination of 5-methylcytosine, and follow-on base excision repair enzymes restore a G⅐C pair. TDG cleaves the N-glycosylic bond of dT and some other nucleotides, including 5-substituted 2-deoxyuridine analogs, once they have been flipped from the helix into its active site. We examined the role of two strictly conserved residues; Asn 140 , implicated in the chemical step, and Arg 275 , implicated in nucleotide flipping. The N140A variant binds substrate DNA with the same tight affinity as wild-type TDG, but it has no detectable base excision activity for a G⅐T substrate, and its excision rate is vastly diminished (by ϳ10 4.4 -fold) for G⅐U, G⅐FU, and G⅐BrU substrates. Thus, Asn 140 does not contribute substantially to substrate binding but is essential for the chemical step, where it stabilizes the transition state by ϳ6 kcal/mol (compared with 11.6 kcal/ mol stabilization provided by TDG overall). Our recent crystal structure revealed that Arg 275 penetrates the DNA minor groove, filling the void created by nucleotide flipping. We found that the R275A and R275L substitutions weaken substrate binding and substantially decrease the base excision rate for G⅐T and G⅐BrU substrates. Our results indicate that Arg 275 promotes and/or stabilizes nucleotide flipping, a role that is most important for target nucleotides that are relatively large (dT and bromodeoxyuridine) and/or have a stable N-glycosylic bond (dT). Arg 275 does not contribute substantially to the binding of TDG to abasic DNA product, and it cannot account for the slow product release exhibited by TDG.The chemically reactive bases in DNA are continuously modified by oxidation, alkylation, and deamination, creating mutagenic and cytotoxic lesions that are implicated in aging and diseases including cancer (1, 2). Such damage is handled predominantly by the base excision repair pathway, initiated by one of many damage-specific DNA glycosylases (3). These enzymes use a nucleotide flipping mechanism to find damaged bases within the vast excess of normal DNA and cleave the base-sugar (N-glycosylic) bond to release the base, and follow-on base excision repair proteins complete the repair process.Thymine DNA glycosylase (TDG) 2 removes thymine from mutagenic G⅐T mispairs, one of the few glycosylases that removes a normal base from DNA. Consistent with the need to avoid acting upon undamaged DNA, TDG activity is 18,000-fold greater for G⅐T mispairs relative to A⅐T pairs (4). TDG is also specific for a particular DNA sequence, exhibiting the highest activity for G⅐T mispairs (and other lesions) with a

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Toward a Detailed Understanding of Base Excision Repair Enzymes: Transition State and Mechanistic Analyses of N-Glycoside Hydrolysis and N-Glycoside Transfer

Cited by 238 publications

References 507 publications

Physical and Functional Interactions between Uracil-DNA Glycosylase and Proliferating Cell Nuclear Antigen from the Euryarchaeon Pyrococcus furiosus

Physical and Functional Interactions between Uracil-DNA Glycosylase and Proliferating Cell Nuclear Antigen from the Euryarchaeon Pyrococcus furiosus

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

Role of Two Strictly Conserved Residues in Nucleotide Flipping and N-Glycosylic Bond Cleavage by Human Thymine DNA Glycosylase

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