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.

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.

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).

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.

(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.