Functional characterization of the novel DNA repair gene MBD4
Wong, Edmund Hong-Sing
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Mbd4 (methyl-CpG binding domain 4) is a novel mammalian repair enzyme that was first identified as a member of a family of proteins containing a distinct domain that binds to methyl-CpG dinucleotides. In addition, the human protein was shown to interact with the DNA mismatch repair protein MLH1. Mutations in Mbd4 have been observed in human colorectal carcinomas exhibiting microsatellite instability (MSI), a hallmark of mismatch repair (MMR) deficiency. Recent biochemical studies have advanced the understanding that Mbd4 is a glycosylase involved in the repair of mismatched G-T and G-U residues at methylated CpG sites. These results taken together suggest that MBD4 defects might confer cancer predisposition.;To clarify the role of Mbd4 in DNA repair in vivo and to examine the impact of Mbd4 inactivation on gastrointestinal (GI) tumorigenesis, we introduced a null mutation into the murine Mbd4 gene by gene-targeting. Heterozygous and homozygous Mbd4 mutant mice develop normally and do not show increased cancer susceptibility or reduced survival. Because of the speculative role of Mbd4 in mismatch repair, we assessed the mutation frequency at microsatellite sequences in the mouse genome. In one mononucleotide marker and two dinucleotide markers examined, Mbd4 inactivation did not increase MSI. Furthermore, we found that MMR activity, as detected by an in vitro assay system, was not compromised in Mbd4-deficient cellular extracts. Our findings support the notion that Mbd4 does not have a direct role in MMR.;Although Mbd4 inactivation did not increase the mutation frequency at microsatellite sequences, it did result in a two to three-fold increase in C → T transition mutations at CpG sites within the lacI mutational reporter of splenocytes and small intestinal epithelial cells. The combination of Mbd4-deficiency with a germ line mutation in the adenomatous polyposis coli (Apc) gene increased the tumor number in the GI tract and accelerated tumor progression. Most dramatically, the number of microadenomatous lesions was 10-fold higher in Mbd4 -/-/Apc1638N /+ mice than in Apc1638N/+ single mutants. The change in the GI cancer phenotype was associated with an increase in somatic C → T mutations at CpG sites within the coding region of the wild type Apc allele, a finding in remarkable agreement with our lacI analysis and the previously ascribed biochemical activity of Mbd4. These studies indicate that although inactivation of Mbd4 does not by itself cause cancer predisposition in mice, can alter the mutation spectrum in cancer cells and modify the cancer predisposition phenotype.
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