Incision at DNA G.cntdot.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.cntdot.T binding reaction

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.The integrity of genetic information is constantly challenged by a variety of endogenous and exogenous DNA damaging agents (1-3). Cellular DNA transactions occur in aqueous solution containing reactive oxygen species, and, as such, DNA is prone to both hydrolytic and oxidative damage. Hydrolysis of the N-glycosyl bond yields apurinic and, less frequently, apyrimidinic sites that are highly mutagenic. Hydrolytic deamination of cytosine and 5-methylcytosine (M) 1 generates G:U and G:T mismatches, respectively. Oxidative lesions include 8-oxoguanine, thymine glycol, and formamidopyrimidine derivatives of adenine and guanine (1, 2). In addition to endogenous damaging processes, DNA is exposed to the attack of exogenous reactive species, including alkylating agents and the carcinogens vinyl chloride and ethyl carbamate. Alkylating agents primarily alkylate the N 3 position of purines and the N 7 and O 6 positions of guanine (1, 2), whereas metabolites of vinyl chloride and ethyl carbamate generate cyclic (etheno) DNA adducts, such as 3,N 4 -ethenocytosine, 1,N 6 -ethenoadenine, 1,N 2 -ethenoguanine and N 2 ,3-ethenoguanine (4, 5). Efficient correction of these DNA lesions relies on the action of several enzymes belonging to the base excision repair system (2, 6 -9). Unlike nucleotide excision repair or long-patch mismatch repair (MMR), base excision repair enzymes usually act in a lesion-specific fashion on a single damaged or mismatched nucleotide. Given the mutagenic potential of DNA lesions, continuing elucidation of the biochemical activities, damage spectrum and specificity of base excision repair enzymes has direct implications on cancer and aging (2).In an effort to isolate new human proteins involved in DNA repair, ...

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“…context of a CpG site regardless of its methylation status (34,(45)(46)(47), lacks an MBD domain altogether.…”

Section: ϫ6mentioning

confidence: 99%

Biphasic Kinetics of the Human DNA Repair Protein MED1 (MBD4), a Mismatch-specific DNA N-Glycosylase

Petronzelli¹,

Riccio²,

Markham³

et al. 2000

Journal of Biological Chemistry

160

161

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“…20,21 However, when sequence-context effects are taken into account, the preferred substrate for Tdg is a T·G mismatch in a methylated or unmethylated CpG dinucleotide. 19,[22][23][24] Methyl-CpG binding domain protein 4 (Mbd4) is another DNA glycosylase implicated in the suppression of CpG mutation. MBD4 can bind methylated CpG sites through its N-terminal methyl-binding domain and has been demonstrated to excise thymine residues from T·G mismatches.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Dnmt3b:Thymine-DNA Glycosylase Interaction and Stimulation of Thymine Glycosylase-Mediated Repair by DNA Methyltransferase(s) and RNA

Boland

Christman

2008

Journal of Molecular Biology

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SummaryMethylation of cytosine residues in CpG dinucleotides plays an important role in epigenetic regulation of gene expression and chromatin structure/stability in higher eukaryotes. DNA methylation patterns are established and maintained at CpG dinucleotides by DNA methyltransferases (Dnmt1 and Dnmts 3a and 3b). In mammals and many other eukaryotes, the CpG dinucleotide is underrepresented in the genome. This loss is postulated to be the result of unrepaired deamination of cytosine (C) and 5-methylcytosine (5mC) to uracil (U) and thymine (T), respectively. Two thymine glycosylases are believed to reduce the impact of 5mC deamination. G/T mismatchspecific thymine-DNA glycosylase (Tdg) and methyl-CpG binding domain protein 4 (Mbd4) can both excise uracil or thymine at U·G and T·G mismatches to initiate base excision repair. Here, we report the characterization of interactions between Dnmt3b and both Tdg and Mbd4. Our results demonstrate that both Tdg and Dnmt3b are colocalized to heterochromatin and also demonstrate reduction of T·G mismatch repair efficiency upon loss of DNA methyltransferase expression as well as a requirement for an RNA component for correct T·G mismatch repair.

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“…Thus Day and co-workers (Sibghat-Ullah and Day, 1992) and Karran and colleagues (Griffin et al, 1994) showed that the enzyme present in crude extracts processed also 6-O-methylguanine/thymine mispairs by excising the thymine. The same workers also showed that the enzyme is sensitive to the sequence context of the mispair (Griffin and Karran, 1993;Day, 1993, 1995). Given the many capabilities of the enzyme in vitro, it is not easy to state with any certainty which, if any, of these enzymatic reactions reflect the true physiological function of the protein.…”

mentioning

confidence: 97%

Cloning and Expression of Human G/T Mismatch-specific Thymine-DNA Glycosylase

Neddermann¹,

Gallinari²,

Lettieri³

et al. 1996

Journal of Biological Chemistry

253

175

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Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of baseexcision repair enzymes.

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Incision at DNA G.cntdot.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.cntdot.T binding reaction

Cited by 30 publications

References 27 publications

Biphasic Kinetics of the Human DNA Repair Protein MED1 (MBD4), a Mismatch-specific DNA N-Glycosylase

Biphasic Kinetics of the Human DNA Repair Protein MED1 (MBD4), a Mismatch-specific DNA N-Glycosylase

Characterization of Dnmt3b:Thymine-DNA Glycosylase Interaction and Stimulation of Thymine Glycosylase-Mediated Repair by DNA Methyltransferase(s) and RNA

Cloning and Expression of Human G/T Mismatch-specific Thymine-DNA Glycosylase

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