Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Mar 30;96(7):3969-74.
doi: 10.1073/pnas.96.7.3969.

MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1

A Bellacosa  1 , L Cicchillitti, F Schepis, A Riccio, A T Yeung, Y Matsumoto, E A Golemis, M Genuardi, G Neri
Affiliations

MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1

A Bellacosa et al. Proc Natl Acad Sci U S A. .

Abstract

The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Interaction between f5/MED1 and MLH1. (A) Specific association of f5 and MLH1 by yeast interaction trap. EGY191 cells were cotransformed with various combinations of plasmids, as indicated, along with the lacZ reporter pSH18–34. Individual transformants were replated onto Leu (−)/galactose plates to score for activation of the LEU2 reporter (Left) and onto X-Gal/galactose plates to score for activation of the lacZ reporter (Right). All interactions were galactose-specific. K-rev-1 and Krit1 represent a positive control for interaction. (B) Coimmunoprecipitation of MED1 and MLH1 from human cells. A band reacting with the anti-MLH1 antibody and comigrating with MLH1 is detected by Western blotting in antihemagglutinin immunoprecipitates from HT-MED1/CMV5-transfected HEK-293 cells and not from CMV5-transfected control cells (Upper). Western blotting of a parallel gel with the antihemagglutinin antibody confirms expression of the HT-MED1 construct in transfected HEK-293 cells (Lower). Lysis buffers contained 0.5% Nonidet P-40 (lanes 1–4), 0.2% Nonidet P-40 (lanes 5 and 6), or 1% Triton X-100 (lanes 7 and 8).
Figure 2
Figure 2
Sequence and domain organization of MED1. Predicted amino acid sequence (A) and schematic structure (B) of human MED1; the MBD and the endonuclease domain (endo) are enclosed in the gray and white box, respectively. Putative nuclear localization signals are underlined. The bar depicts the sequence of the original f5 clone. (C) Homology of the MBD region of MED1 to the MBD of the rat protein MeCP2 and the human protein PCM1. (D) Comparison of the endonuclease region of MED1 with related sequences from M. thermoautotrophicum mismatch glycosylase (Mig.Mth), E. coli endonuclease III (EndoIII), M. luteus UV endonuclease (UV-Endo), and E. coli MutY. Identical amino acids are boxed; similar amino acids are shaded.
Figure 3
Figure 3
Mobility-shift assay of MED1 MBD with fully methylated, hemimethylated, and unmethylated DNA probes. The MED1 MBD binds to 32P-labeled double-stranded oligonucleotides containing four fully methylated (lane 2) or hemimethylated CpG sites (lane 7). No binding is detected when the unmethylated probe is used (lane 12). For binding competition, the cold oligonucleotides used are indicated.
Figure 4
Figure 4
Endonuclease activity of MED1. (A) The indicated increasing amounts of wild-type MED1 (wt) and a deletion mutant lacking the endonuclease domain (Δendo) were incubated with 0.25 μg of supercoiled pCR2 plasmid at 37°C for 30 min. Reaction products were analyzed by agarose gel electrophoresis (Upper). The migration of nicked, linearized, and supercoiled DNA is indicated. A small amount of nicked molecules is present in the plasmid DNA substrate at time zero. M, λ/HindIII digest size standards. (Lower) Densitometric analysis of the nicked (circles), linearized (squares), and supercoiled (triangles) bands. OD, in thousands of units. (B) Time course of MED1 endonuclease activity. MED1 wt and Δendo (12.5 ng, approximately 170 fmol) were incubated with 1.5 μg (approximately 580 fmol) of pCR2 plasmid at 37°C; at the indicated time points, an aliquot of the reaction was removed for electrophoretic analysis. (C) Endonuclease activity of the recombinant MED1 endonuclease domain (codons 455–580, endo). Endo (3.6 ng, approximately 170 fmol) was incubated with 1.5 μg (approximately 580 fmol) of pCR2 plasmid at 37°C and processed as in B.
Figure 5
Figure 5
(A) In situ β-gal staining of pooled cultures of SW480 cl.1 cells stably transfected with empty vector, wild-type MED1 (wt), or deletion mutants (ΔMBD and Δendo) and subsequently transfected with β-gal reporter genes containing an out-of-frame (pCAR-OF, Upper) or in-frame (pCAR-IF, Lower) (CA) microsatellite repeat. Representative fields of a triplicate experiment are shown. β-gal staining is approximately equal in the pCAR-IF-transfected cell lines expressing different MED1 constructs, whereas, among the pCAR-OF-transfected cell lines, only the ΔMBD-expressing culture contains many blue cells. (B) Relative β-gal staining of mass cultures of SW480 cells stably transfected with empty vector, wild-type MED1 (wt), or deletion mutants (ΔMBD and Δendo). Relative β-gal staining represents the ratio between the percentage of blue area in optical fields of pCAR-OF- and pCAR-IF-transfected cultures. Data are the mean ± SD derived from the analysis of 10 optical fields for each culture.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

  • MBD4 loss results in global reactivation of promoters and retroelements with low methylated CpG density.
    Papin C, Ibrahim A, Sabir JSM, Le Gras S, Stoll I, Albiheyri RS, Zari AT, Bahieldin A, Bellacosa A, Bronner C, Hamiche A. Papin C, et al. J Exp Clin Cancer Res. 2023 Nov 14;42(1):301. doi: 10.1186/s13046-023-02882-z. J Exp Clin Cancer Res. 2023. PMID: 37957685 Free PMC article.
  • DNA demethylation by TDG.
    Dalton SR, Bellacosa A. Dalton SR, et al. Epigenomics. 2012 Aug;4(4):459-67. doi: 10.2217/epi.12.36. Epigenomics. 2012. PMID: 22920184 Free PMC article. Review.
  • Noncatalytic Domains in DNA Glycosylases.
    Torgasheva NA, Diatlova EA, Grin IR, Endutkin AV, Mechetin GV, Vokhtantsev IP, Yudkina AV, Zharkov DO. Torgasheva NA, et al. Int J Mol Sci. 2022 Jun 30;23(13):7286. doi: 10.3390/ijms23137286. Int J Mol Sci. 2022. PMID: 35806289 Free PMC article. Review.
  • MBD4 interacts with and recruits USP7 to heterochromatic foci.
    Meng H, Harrison DJ, Meehan RR. Meng H, et al. J Cell Biochem. 2015 Mar;116(3):476-85. doi: 10.1002/jcb.25001. J Cell Biochem. 2015. PMID: 25358258 Free PMC article.
  • Integrated mutational landscape analysis of poorly differentiated high-grade neuroendocrine carcinoma of the uterine cervix.
    Bellone S, Jeong K, Halle MK, Krakstad C, McNamara B, Greenman M, Mutlu L, Demirkiran C, Hartwich TMP, Yang-Hartwich Y, Zipponi M, Buza N, Hui P, Raspagliesi F, Lopez S, Paolini B, Milione M, Perrone E, Scambia G, Altwerger G, Ravaggi A, Bignotti E, Huang GS, Andikyan V, Clark M, Ratner E, Azodi M, Schwartz PE, Quick CM, Angioli R, Terranova C, Zaidi S, Nandi S, Alexandrov LB, Siegel ER, Choi J, Schlessinger J, Santin AD. Bellone S, et al. Proc Natl Acad Sci U S A. 2024 Apr 23;121(17):e2321898121. doi: 10.1073/pnas.2321898121. Epub 2024 Apr 16. Proc Natl Acad Sci U S A. 2024. PMID: 38625939 Free PMC article.
See all "Cited by" articles

References

    1. Modrich P. Science. 1994;266:1959–1960. - PubMed
    1. Fishel R, Kolodner R D. Curr Opin Genet Dev. 1995;5:382–395. - PubMed
    1. Modrich P, Lahue R. Annu Rev Biochem. 1996;65:101–133. - PubMed
    1. Jiricny J. EMBO J. 1998;17:6427–6436. - PMC - PubMed
    1. Lynch H T, Smyrk T. Cancer. 1996;78:1149–1167. - PubMed

Publication types

MeSH terms

Associated data