Investigation of the substrate spectrum of the human mismatch-specific DNAN-glycosylase MED1 (MBD4): Fundamental role of the catalytic domain

The human DNA repair protein MED1 (also known as MBD4) was isolated as an i nteractor of the mismatch repair protein MLH1 in a yeast two-hybrid screeni ng. MED1 has a tripartite structure with an N-terminal 5-methylcytosine bin ding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. Indeed, MED 1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil paired with guanine. The glycosylase activity of MED1 pr eferentially targets G:T mismatches in the context of CpG sites; this indic ates that MED1 is involved in the repair of deaminated 5-methylcytosine. In terestingly, frameshift mutations of the MED1 gene have been reported in hu man colorectal, endometrial, and pancreatic cancers. For its putative role in maintaining genomic fidelity at CpG sites, it is important to characteri ze the biochemical properties and the substrate spectrum of MED1. Here we s how that MED1 works under a wide range of temperature and pH, and has a lim ited optimum range of ionic strength. MED1 has a weak glycosylase activity on the mutagenic adduct 3,N-4-ethenocytosine, a metabolite of vinyl chlorid e and ethyl carbamate. The differences in glycosylase activity on G:U and G :T substrates are not related to differences in substrate binding and likel y result from intrinsic differences in the chemical step. Finally, the isol ated catalytic domain of MED1 retains the preference for G:T and G:U substr ates 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. J. Cell. Physiol. 185:473-480, 2000. (C) 2000 Wiley-Liss, Inc.