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

Abstract

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, ...

A human cancer-associated truncation of MBD4 causes dominant negative impairment of DNA repair in colon cancer cells

“…The first report of substrates of MBD4 showed preference for T Á G products of methyl-C deamination within methyl-CpG sites . Later reports demonstrated a larger set of in vitro CpG mismatches for MBD4 recognition, such as U Á G (Petronzelli et al, 2000a, b), 5-fluorouracil (5-FU) Á G (Petronzelli et al, 2000b), ethenecytosine (eC) Á G (Petronzelli et al, 2000a), 5-formyluracil (5-FoU) Á G (Liu et al, 2002), T Á O 6 -methyl-guanine (O 6 -meG) (Cortellino et al, 2003) and thymine glycol (Tg) Á G (Yoon et al, 2003). Total absence of MBD4 in mice caused an increase in MF especially at CpG sites (Millar et al, 2002;Wong et al, 2002).…”

Methyl CpG binding proteins: coupling chromatin architecture to gene regulation

“…Recombinant MBD4 induces nicks and linearization of supercoiled plasmids although the protein lacks endonuclease activity on oligonucleotide substrates (Hendrich et al, 1999b). MBD4 clearly has speci®c DNA N-glycosylase activity with a strong preference for G : T mismatches (Hendrich et al, 1999b;Petronzelli et al, 2000). Surprisingly, the MBD domain does not appear to in¯uence the speci®city of the glycosylase activity for G : T mismatches (Hendrich et al, 1999b;Petronzelli et al, 2000).…”

“…MBD4 clearly has speci®c DNA N-glycosylase activity with a strong preference for G : T mismatches (Hendrich et al, 1999b;Petronzelli et al, 2000). Surprisingly, the MBD domain does not appear to in¯uence the speci®city of the glycosylase activity for G : T mismatches (Hendrich et al, 1999b;Petronzelli et al, 2000). However, while the MBD domain binds to symmetrically methylated CpG dinucleotides, it prefers 5-methyl CpG paired with TpG (Hendrich et al, 1999b).…”

“…However, while the MBD domain binds to symmetrically methylated CpG dinucleotides, it prefers 5-methyl CpG paired with TpG (Hendrich et al, 1999b). As this particular G : T mismatch is the expected product of deamination of a single 5-methyl C in a symmetrically methylated CpG dinucleotide, MBD4 has been designated a repair enzyme speci®c for methylated DNA (Hendrich et al, 1999b;Petronzelli et al, 2000). Interestingly, mutations in the MBD4 gene have been isolated in carcinomas with microsatellite instability, where DNA mismatch repair activity is defective (Bader et al, 1999;Riccio et al, 1999).…”

Uracil in DNA – occurrence, consequences and repair

“…Some members of the HhH -GPD superfamily, including MBD4, also contain a methyl-CpG binding domain (MBD) which has an alpha beta 2-layer sandwich architecture. It has been shown that MBD4 acts as a G : T and G : U specific thymine and uracil glycosylase (Hendrich et al, 1999;Petronzelli et al, 2000). The 3D structure for MBD4 is not known.…”