Investigation of the substrate spectrum of the human mismatch‐specific DNA N ‐glycosylase MED1 (MBD4): Fundamental role of the catalytic domain

Abstract

The human DNA repair protein MED1 (also known as MBD4) was isolated as an interactor of the mismatch repair protein MLH1 in a yeast two-hybrid screening. MED1 has a tripartite structure with an N-terminal 5-methylcytosine binding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. Indeed, MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil paired with guanine. The glycosylase activity of MED1 preferentially targets G:T mismatches in the context of CpG sites; this indicates that MED1 is involved in the repair of deaminated 5-methylcytosine. Interestingly, frameshift mutations of the MED1 gene have been reported in human colorectal, endometrial, and pancreatic cancers. For its putative role in maintaining genomic fidelity at CpG sites, it is important to characterize the biochemical properties and the substrate spectrum of MED1. Here we show that MED1 works under a wide range of temperature and pH, and has a limited optimum range of ionic strength. MED1 has a weak glycosylase activity on the mutagenic adduct 3,N(4)-ethenocytosine, a metabolite of vinyl chloride and ethyl carbamate. The differences in glycosylase activity on G:U and G:T substrates are not related to differences in substrate binding and likely result from intrinsic differences in the chemical step. Finally, the isolated catalytic domain of MED1 retains the preference for G:T and G:U substrates in the context of methylated or unmethylated CpG sites. This suggests that the catalytic domain is fundamental, and the 5-methylcytosine binding domain dispensable, in determining the substrate spectrum of MED1.

Hijacking of the Human Alkyl-N-purine-DNA Glycosylase by 3,N4-Ethenocytosine, a Lipid Peroxidation-induced DNA Adduct

“…The weak inhibition by ⑀A⅐T and I⅐T might be due to the high K m value of ANPG for these substrates (24 and 69 nM, respectively) (41,42). No significant effect on ⑀A-DNA glycosylase activity of ANPG was detected in the presence of C⅐G, G⅐T, G⅐U, THF⅐G, 8-oxoG⅐C, 8-oxoG⅐A, 5ohU⅐G, DHU⅐G, DHT⅐A, and I⅐T oligonucleotides, even at 8-fold molar excess (lanes 3-8, lanes [13][14][15][16], and data not shown). 2-and 20-fold reductions of the incision were observed in the presence of 1-and 8-fold molar excess of ⑀C⅐G, respectively (lanes [11][12], indicating that ⑀C is a more efficient inhibitor than ⑀A (or Hx) (lanes 10 and 12).…”

“…2 A, schematic representation of the 5Ј-flap structure DNA template containing either a C⅐G or a ⑀C⅐G base pair and possible elongation products: 41-mer, full-sized product; 25-mer, ⑀C termination product; 20-mer, ANPG⅐⑀C termination product; 13-mer, 5Ј-32 P-labeled primer. B, 10 nM 5Ј-32 P-labeled C⅐G (lanes 1-8) and/or ⑀C⅐G (lanes 9 -16) primer/ template were incubated with (lanes 3-8 and lanes [11][12][13][14][15][16] or without (lanes 1-2 and lanes 9 -10) 1 unit of Klenow fragment, and the primer extension reaction was performed for 5 min at 37°C. Lanes 1 and 9, no enzyme; lanes 2 and 10, 500 nM ANPG80; lanes 3 and 11, 10 nM ANPG80; lanes 4 and 12, 20 nM ANPG80; lanes 5 and 13, 50 nM ANPG80; lanes 6 and 14, 100 nM ANPG80; lanes 7 and 15, 200 nM ANPG80; lanes 8 and 16, 500 nM ANPG80.…”

“…However, excision efficiency of ⑀C by hTDG is rather poor (12). Recently, two additional enzymes that excise ⑀C, have been identified in human cells: the methyl-CpG binding domain protein (MBD4/MED1) (14) and single-strand monofunctional uracil-DNA glycosylase (SMUG1) (15). ¶ To whom correspondence should be addressed: Tel.…”

Mutational spectra of aflatoxin B 1 in vivo establish biomarkers of exposure for human hepatocellular carcinoma

“…To date, in vitro studies have provided contradictory evidence as to whether the presence of a 5-methylcytosine adjacent to a guanine enhances reactivity toward the AFB 1 -epoxide (33,34). It is also possible that an AFB 1 adduct, once formed in a methylated CpG site, is refractory to repair, because methylated sites are often bound by regulatory proteins (e.g., MBD4) (35). Taken together, the hot and cold spots for mutation identified in this work will guide the design of further experiments that will provide mechanistic insights into the pathways by which AFB 1 adducts site-specifically form and are removed in vivo.…”

Mbd4 inactivation increases C→T transition mutations and promotes gastrointestinal tumor formation

“…However, analysis of the Mbd4 amino acid sequence revealed that it contained a C-terminal catalytic domain that shares high homology to many known bacterial DNA glycosylases and lyases. Further biochemical analysis using oligonucleotide substrates showed that Mbd4 is a DNA glycosylase that could act in the repair of G-T or G-U mismatches at CpG sites resulting from deamination of 5-methyl cytosine or cytosine, respectively (7,8).…”