A human cancer-associated truncation of MBD4 causes dominant negative impairment of DNA repair in colon cancer cells

Abstract

MBD4 binds to methylated DNA and acts as a thymine DNA glycosylase in base excision repair. Deficiency of MBD4 in mice enhances mutation at CpG sites and alters apoptosis in response to DNA damage, but does not increase tumorigenesis in mismatch repair-deficient mice. However, in humans, frameshift mutation of MBD4, rather than deletion, is what occurs in up to 43% of microsatellite unstable colon cancers. There is no murine equivalent of this mutation. We now show that recombinant truncated MBD4 (MBD4 tru ) inhibits glycosylase activities of normal MBD4 or Uracil DNA glycosylase in cell-free assays as a dominant negative effect. Furthermore, overexpression of MBD4 tru in Big Blue (lacI)-transfected, MSI human colorectal carcinoma cells doubled mutation frequency, indicating that the modest dominant negative effect on DNA repair can occur in living cells in short-term experiments. Intriguingly, the whole mutation spectrum was increased, not only at CpG sites, suggesting that truncated MBD4 has a more widespread effect on genomic stability. This demonstration of a dominant negative effect may be of significance in tumour progression and acquisition of drug resistance.

Modulation of the activity of methyl binding domain protein 4 (MBD4/MED1) while processing iododeoxyuridine generated DNA mispairs

“…Interestingly, the inhibitory action of MBD was observed in both a cis-and trans-manner. This observation is different from a previously published study, 14 possibly because of a difference in the composition of the MBD4 tru and/or the substrate. However, our data are supported by a more recently published study which demonstrated that the efficiency of MED1 glycosylase activity was partially dependent on the extent of methylation of the DNA substrate.…”

“…A MBD containing truncated MED1 protein has been postulated to act in a dominant negative fashion to inhibit the activity of full length MED1. 14 We studied this effect on methylated G:IU substrate using equimolar concentrations of recombinant purified MBD the action of the GD on G:IU mispairs located in unmethylated, hemimethylated and fully methylated contexts. 5 In the current study, we measured the relative activity of full length MED1 on G:IU mispairs (Fig.…”

MBD4 Facilitates Immunoglobulin Class Switch Recombination

“…Truncated Mbd4 proteins lack dominant negative effects. Because truncated MBD4 protein arising from residual expression of exons 1 to 3 or exons 1 to 5 could potentially act as dominant negative mutants (33) and inhibit CSR, we analyzed the pattern of Mbd4 transcription in control and Mbd4 KO cells. Although microarray analyses indicate that the transcript abundance of Mbd4 exons 1 to 3 is invariant, longer transcripts are severely reduced, beginning at exon 4, for the Mbd4 KO relative to controls ( Fig.…”

Tagging single nucleotide polymorphisms in MBD4 are associated with risk of lung cancer in a Chinese population

“…In vivo experiments also indicated that MBD4 can mediate the cellular response to DNA damage [19][20][21] and act as a candidate tumor suppressor gene [7].…”