The DNAN-Glycosylase MED1 Exhibits Preference for Halogenated Pyrimidines and Is Involved in the Cytotoxicity of 5-Iododeoxyuridine

Abstract

The base excision repair protein MED1 (also known as MBD4), an interactor with the mismatch repair protein MLH1, has a central role in the maintenance of genomic stability with dual functions in DNA damage response and repair. MED1 acts as a thymine and uracil DNA N-glycosylase on T:G and U:G mismatches that occur at cytosine-phosphate-guanine (CpG) methylation sites due to spontaneous deamination of 5-methylcytosine and cytosine, respectively. To elucidate the mechanisms that underlie sequence discrimination by MED1, we did single-turnover kinetics with the isolated, recombinant glycosylase domain of MED1. Quantification of MED1 substrate hierarchy confirmed MED1 preference for mismatches within a CpG context and showed preference for hemimethylated base mismatches. Furthermore, the k st values obtained with the uracil analogues 5-fluorouracil and 5-iodouracil were over 20-to 30-fold higher than those obtained with uracil, indicating substantially higher affinity for halogenated bases. A 5-iodouracil precursor is the halogenated nucleotide 5-iododeoxyuridine (5IdU), a cytotoxic and radiosensitizing agent. Cultures of mouse embryo fibroblasts (MEF) with different Med1 genotype derived from mice with targeted inactivation of the gene were evaluated for sensitivity to 5IdU. The results revealed that Med1-null MEFs are more sensitive to 5IdU than wild-type MEFs in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Furthermore, high-performance liquid chromatography analyses revealed that Med1-null cells exhibit increased levels of 5IdU in their DNA due to increased incorporation or reduced removal. These findings establish MED1 as a bona fide repair activity for the removal of halogenated bases and indicate that MED1 may play a significant role in 5IdU cytotoxicity. (Cancer Res 2006; 66(15): 7686-93)

MBD4-Mediated Glycosylase Activity on a Chromatin Template Is Enhanced by Acetylation

“…5A and 8C), in agreement with results reported previously for hemimethylation immediately adjacent to the mismatch (52). Also, the enzymatic activity of the glycosylase domain of MBD4 itself has been shown, in some previous studies, to prefer T/G mismatches within the context of a CpG site when the site is hemimethylated (65). In the study by Turner and coworkers, the single-turnover rate of the enzyme was found to be 17% higher than that in the nonmethylated context (65).…”

“…Surprisingly, the activity of the glycosylase is likewise unaffected by the presence of the methyl-binding domain (51). Despite the aforementioned preference of the enzyme for methylase recognition sites, there is some disagreement about whether the activity of the intact protein is affected by the presence or absence of a methyl group on the DNA strand opposite the mismatched T (52,65,66).…”

Effect of UVB irradiation on microRNA expression in mouse epidermis

“…MED1 acts as a thymine and uracil DNA N-glycosylase on T:G and U:G mismatches that occur at cytosine-phosphate-guanine (CpG) methylation sites due to the spontaneous deamination of 5-methylcytosine and cytosine, respectively. This event indicates that MED1 is involved in the removal of methylated DNA (12). Abnormal DNA methylation (including hypermethylation and hypomethylation) is a hallmark of the majority of cancers, including colon, lung, prostate and breast cancers, and contributes to carcinogenesis by silencing tumor suppressor genes, upregulating oncogenes and/or reducing genomic stability (13,14).…”

Silent beginning: Early silencing of the MED1/MBD4 gene in colorectal tumorigenesis

“…In particular, MED1 removes Ts mispaired with those Gs that are methylated by these drugs ( 6 O-methyl-guanine), suggesting that MED1 may be involved in the resistance to these drugs. 10,11 In fact, Cortellino et al and Sansom et al demonstrated that a normal MED1 is required for apoptosis of cells damaged by the mutagenic effect of several other drugs, including methylating agents. 10,12 In MED1-deficient cells, the DNA damage accumulates without the induction of cell cycle arrest and apoptosis.…”