Investigation of the substrate spectrum of the human mismatch-specific DNA N-glycosylase MED1 (MBD4): fundamental role of the catalytic domain.
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.
Petronzelli
F
F
Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA.
Riccio
A
A
Markham
G D
GD
Seeholzer
S H
SH
Genuardi
M
M
Karbowski
M
M
Yeung
A T
AT
Matsumoto
Y
Y
Bellacosa
A
A
D020137:Base Pair Mismatch; D001665:Binding Sites; D002384:Catalysis; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D013379:Substrate Specificity
D004706:Endodeoxyribonucleases; C118712:MBD4 protein, human