The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites

Hendrich, Brian; Hardeland, Ulrike; Ng, Huck‐Hui; Jiricny, Josef; Bird, Adrian

doi:10.1038/45843

Cited by 580 publications

(469 citation statements)

References 24 publications

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“…Briefly, 10 ng recombinant wtMBD4 or 0.2 U uracil DNA glycosylase (New England Biolabs, Hertfordshire, UK) protein was incubated with annealed oligonucleotides (the reverse strand of each pair having been endlabelled with [ 33 P] ATP) with or without recombinant MBD4 tru in a constant total volume of 40 ml. Primer combinations (Hendrich and Bird, 1998;Hendrich et al, 1999) used were BH:BHrev, BH:BHrevU, MM2:MM3revU.…”

Section: Glycosylase Assaysmentioning

confidence: 99%

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

2007

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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.

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Section: Glycosylase Assaysmentioning

confidence: 99%

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

2007

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“…In our opinion, these are important studies to perform, because C to T transitions at methylated CpG sites are the most common mutations found in human tumors and genetic disease. 29 TDG and MBD4 function in the repair of deaminated cytosine. Deamination of 5-methycytosine (5-meC) to thymine is a common event that gives rise to the T:G mispair.…”

Section: Germline and Tumor-associated Variants Of Dna Glycosylases Mmentioning

confidence: 99%

“…In fact, the mutation rate of 5-methylcytosine to thymine is 10-to 50-fold higher than other transition mutations in humans. [29][30][31] The TDG and MBD4 DNA glycosylases excise thymine from T:G mispairs. TDG is also able to excise adducts such as ethenocytosine, that arise as a byproduct of lipid peroxidation.…”

Section: Germline and Tumor-associated Variants Of Dna Glycosylases Mmentioning

confidence: 99%

Is Base Excision Repair a Tumor Suppressor Mechanism?

Sweasy¹,

Lang²,

DiMaio³

2006

Cell Cycle

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The base excision repair pathway is critical for the removal of oxidized and methylated bases from the DNA. Much of this DNA damage arises endogenously, as a result of oxygen metabolism. Several proteins including DNA glycosylases, the APE1 endonuclease, DNA polymerase β and DNA ligase, act in a highly regulated and coordinated manner during base excision repair to excise the base adducts from the DNA and restore the normal DNA sequence. Both germline and tumor-associated variants of genes encoding these proteins have been identified in humans. In many cases, the protein variant has been shown to have properties that could contribute to the development of cancer, suggesting that base excision repair acts as a tumor suppressor mechanism in humans. Limited epidemiological studies are consistent with this view. Our review of the literature indicates that additional laboratory and epidemiological studies of the role of base excision repair in cancer etiology is warranted.

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“…All forms of UNG examined remove uracil opposite A or G in doublestranded DNA as well as from single-stranded DNA [3,5]. At least four different human nuclear DNA glycosylases, UNG2, single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), thymine-DNA glycosylase (TDG) and methyl-CpG binding domain protein 4 (MBD4, also known as MED1) have been reported to removal uracil from U·G mispairs [6][7][8], and it has not yet been determined whether the different uracil-DNA glycosylases have distinct or overlapping physiological functions. However, the recent experiments by Akbari and co-workers indicate that UNG2 is the major activity to initiate base excision repair of deaminated cytosine (U·G) and perhaps the only enzyme to remove misincorporated uracil (U·A) [6].…”

Section: Introductionmentioning

confidence: 99%

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Bennett

2005

DNA Repair

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Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl 2 . The functional significance of the UNG2·PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-3 H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl 2 . The stimulatory effect of PCNA was increased by the addition of MgCl 2 ; however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl 2 and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.

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The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites

Cited by 580 publications

References 24 publications

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

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

Is Base Excision Repair a Tumor Suppressor Mechanism?

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

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