The DNA N-glycosylase MED1 exhibits preference for halogenated pyrimidines and is involved in the cytotoxicity of 5-iododeoxyuridine

David P. Turner, Salvatore Cortellino, Jane E. Schupp, Elena Caretti, Tamalette Loh, Timothy J. Kinsella, Alfonso Bellacosa

Research output: Contribution to journalArticlepeer-review

54 Scopus citations

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 kst 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.

Original languageEnglish
Pages (from-to)7686-7693
Number of pages8
JournalCancer Research
Volume66
Issue number15
DOIs
StatePublished - Aug 1 2006

Keywords

  • Animals
  • Base Pair Mismatch
  • Cell Line
  • CpG Islands
  • Embryo, Mammalian
  • Endodeoxyribonucleases/genetics
  • Gene Silencing
  • Humans
  • Idoxuridine/metabolism
  • Mice
  • Pyrimidines/metabolism
  • Substrate Specificity

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