Kinetics of the Action of Thymine DNA Glycosylase

Waters, T.P.; Swann, Peter F.

doi:10.1074/jbc.273.32.20007

Cited by 141 publications

(187 citation statements)

References 31 publications

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“…The nature of this inhibition is unclear, since Co 2+ and/or Zn 2+ were reported to stimulate the activity of DNA repair enzymes such as E. coli Nfo [54] and Fpg [55], and ZnSO 4 has been included in the reaction buffer of human thymine-DNA glycosylase [56]. Addition of MgCl 2 to the assay buffer did not alter the circular dichroism spectrum of purified UNG2 (data not shown).…”

Section: Discussionmentioning

confidence: 96%

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Bennett

2005

DNA Repair

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Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl 2 . The functional significance of the UNG2·PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-3 H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl 2 . The stimulatory effect of PCNA was increased by the addition of MgCl 2 ; however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl 2 and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.

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Section: Discussionmentioning

confidence: 96%

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Bennett

2005

DNA Repair

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“…These enzymes face the formidable task of removing a normal base, thymine, from G·T mispairs but not from A·T base pairs. Previous studies show that TDG activity is weak for G·T mispairs compared with most in vitro substrates (e.g., G·U), even though G·T mispairs are an important biological substrate for TDG (8,9,(16)(17)(18)(19). This might reflect a mechanism used by TDG to strike a balance between the requirement for efficient repair of mutagenic G·T lesions and the need for avoiding aberrant removal of T from A·T pairs, which can be mutagenic and cytotoxic (13,20,21).…”

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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“…UNG preferentially recognizes uracil residues in single-stranded DNA and is also active on duplex DNA containing U⅐G mispairs and U⅐A base pairs (17). In contrast, human thymine-DNA glycosylase does not recognize uracil in single-stranded DNA but does excise uracil residues in double-stranded DNA with the following specificity: U⅐G Ͼ U⅐C Ͼ U⅐T Ͼ Ͼ U⅐A (18,19). The human MED1 (also termed MBD4) enzyme acts on U⅐G mispairs in the context of methylated or unmethylated CpG sites, but activity against uracil-containing single-stranded DNA and U⅐A, U⅐C, or U⅐T double-stranded DNA substrates was not detected (20).…”

mentioning

confidence: 99%

Fidelity of Uracil-initiated Base Excision DNA Repair in DNA Polymerase β-Proficient and -Deficient Mouse Embryonic Fibroblast Cell Extracts

Bennett

Sung

Mosbaugh

2001

Journal of Biological Chemistry

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Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase ␤ (؉/؉) and (؊/؊) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U⅐A, U⅐G, and U⅐T targets contained within the lacZ␣ gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase ␤ (؉/؉) and (؊/؊) cellfree extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced (ϳ88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting ϳ20% of the repair events. Addition of p21 peptide or protein to DNA polymerase ␤ (؉/؉) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by ϳ2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U⅐T) DNA was determined to be 5.7-7.2 ؋ 10 ؊4 when using DNA polymerase ␤ (؉/؉) and (؊/؊) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U⅐G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U⅐G target and frequently involved a 1-base deletion or incorporation of dTMP.

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Kinetics of the Action of Thymine DNA Glycosylase

Cited by 141 publications

References 31 publications

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

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

Fidelity of Uracil-initiated Base Excision DNA Repair in DNA Polymerase β-Proficient and -Deficient Mouse Embryonic Fibroblast Cell Extracts

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