Active-Site Clashes Prevent the Human 3-Methyladenine DNA Glycosylase from Improperly Removing Bases

Connor, Ellen E.; Wyatt, Michael D.

doi:10.1016/s1074-5521(02)00215-6

Cited by 31 publications

(60 citation statements)

References 41 publications

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“…4A). N169S is a mutant that enhances DNA cleavage activities (24,33). As expected, ODG activities were detected with this mutant.…”

Section: Resultssupporting

confidence: 74%

“…To rule out the possibility that the observed cleavage activities may come from E. coli expression host contamination, assays were performed with an active site mutant ⌬54-E125Q. This mutant is catalytically inactive because of the change of a proposed general base residue Glu to Gln (24,28,31,32). No DNA cleavage was observed from ⌬54-E125Q, indicating that the ODG activities are intrinsic to hAAG (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, the active site appears to act more to discriminate against the normal purines than to specifically recognize the damaged purines. One key feature of this discrimination in the active site is the potential steric clash between Asn-169 of AAG and the N 2 -amino group of guanine (24,33). The damaged adenine derivatives 3-methylA, ⑀A, and hypoxanthine lack the N 2 -amino group.…”

Section: Resultsmentioning

confidence: 99%

“…DNA Glycosylase Activity Assays-Purification of human AAG proteins was carried out as described previously (23,24). DNA glycosylase cleavage assays were performed at 37°C for 60 min in a 10-l reaction mixture containing 10 nM oligonucleotide substrate, an indicated amount of glycosylase, 20 mM Tris-HCl (pH 7.2), 100 mM KCl, 5 mM EDTA, and 2 mM 2-mercaptoethanol as described previously (22).…”

Section: Methodsmentioning

confidence: 99%

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Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

Hitchcock

Dong

Connor

et al. 2004

Journal of Biological Chemistry

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Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N 1 -nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase ␤, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency ϳ80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxacontaining DNA as well. Indeed, AAG can also remove 1,N 6 -ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.

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“…4A). N169S is a mutant that enhances DNA cleavage activities (24,33). As expected, ODG activities were detected with this mutant.…”

Section: Resultssupporting

confidence: 74%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

Hitchcock

Dong

Connor

et al. 2004

Journal of Biological Chemistry

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“…Previous studies of AAG (alkadenine DNA glycosylase) revealed a mutation (N169S) that gives 33-fold faster excision of G from G·C pairs, but marginally (≤ twofold) increased activity for damaged bases (Hx and εA) (51,52). For UNG, mutations that cause large increases in excision of T from A·T pairs or C from G·C pairs lead to decreased rather than increased activity for the target lesion (uracil) (53).…”

Section: Ala145 and His151 Variants Exhibit Aberrant Activity On A·t mentioning

confidence: 99%

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

Maiti

Noon

MacKerell

et al. 2012

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

168

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DNA base excision repair is essential for maintaining genomic integrity and for active DNA demethylation, a central element of epigenetic regulation. A key player is thymine DNA glycosylase (TDG), which excises thymine from mutagenic G·T mispairs that arise by deamination of 5-methylcytosine (mC). TDG also removes 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC produced by Tet enzymes. Recent studies show that the glycosylase activity of TDG is essential for active DNA demethylation and for embryonic development. Our understanding of how repair enzymes excise modified bases without acting on undamaged DNA remains incomplete, particularly for mismatch glycosylases such as TDG. We solved a crystal structure of TDG (catalytic domain) bound to a substrate analog and characterized active-site residues by mutagenesis, kinetics, and molecular dynamics simulations. The studies reveal how TDG binds and positions the nucleophile (water) and uncover a previously unrecognized catalytic residue (Thr197). Remarkably, mutation of two active-site residues (Ala145 and His151) causes a dramatic enhancement in G·T glycosylase activity but confers even greater increases in the aberrant removal of thymine from normal A·T base pairs. The strict conservation of these residues may reflect a mechanism used to strike a tolerable balance between the requirement for efficient repair of G·T lesions and the need to minimize aberrant action on undamaged DNA, which can be mutagenic and cytotoxic. Such a compromise in G·T activity can account in part for the relatively weak G·T activity of TDG, a trait that could potentially contribute to the hypermutability of CpG sites in cancer and genetic disease. D NA base excision repair (BER) plays an established role in maintaining genomic integrity, and recent studies indicate that BER is also essential for active DNA demethylation, a key element of epigenetic gene regulation (1-3). A central player in both processes is thymine DNA glycosylase (TDG), which initiates BER by excising damaged or modified forms of 5-methylcytosine (mC) that arise at 5′-CpG-3′ sites. TDG was discovered for its ability to selectively remove T from G·T mispairs, mutagenic lesions that can arise from deamination of mC to T (4, 5). TDG also excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), oxidized forms of mC that are generated by Tet enzymes (6, 7). In addition, TDG was shown to be essential for active DNA demethylation and for embryonic development (8, 9), a role that likely involves excision of deaminated or oxidized forms of mC generated by a deaminase or Tet enzyme (7,(10)(11)(12). Thus, the ability of TDG to remove deaminated and oxidized forms of mC is important for genetic and epigenetic integrity.The question of how DNA glycosylases remove modified bases without acting upon the huge excess of undamaged DNA remains largely unresolved, particularly for mismatch glycosylases such as TDG and MBD4 (methyl binding domain IV) (13-15). These enzymes face the formidable task of removing a normal base, t...

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Advances in Understanding the Coupling of DNA Base Modifying Enzymes to Processes Involving Base Excision Repair

Wyatt

2013

Advances in Cancer Research

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Active-Site Clashes Prevent the Human 3-Methyladenine DNA Glycosylase from Improperly Removing Bases

Cited by 31 publications

References 41 publications

Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

Advances in Understanding the Coupling of DNA Base Modifying Enzymes to Processes Involving Base Excision Repair

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