Far-Western analysis of PCNA interactions with MMR proteins and their variants. (A) Increasing amounts (top line) of the purified recombinant proteins listed on the right were spotted onto a nitrocellulose membrane, which then was hybridized with PCNA. The membrane subsequently was probed with the anti-PCNA monoclonal antibody 19F4 (see Materials and Methods). [MSH3 (1–200)] 200 N-terminal amino acid residues of hMSH3 expressed in E. coli; (FEN1) flap endonuclease used as a positive control. (B) hMutSα (lane 1), hMSH6Δ77/hMSH2 (lane 2), and hMutSβ (lane 3) subunits separated by SDS-PAGE and visualized by staining with Coomassie blue. (M) Molecular weight markers (from top to bottom: 200, 116, 97, and 66 kD). (C) The denatured proteins from B were transferred onto a nitrocellulose membrane, which was treated as in A above (see Materials and Methods). A and C are autoradiographs of the membranes, where the protein bands were visualized by ECL (see Materials and Methods).

Colocalization of hMSH6 or hMSH3 and PCNA to replication foci in HeLa cells in vivo. (A) Indirect immunofluorescence of HeLa nuclei stained with antibodies against hMSH6 (green) or PCNA (red). Superimposition of the red and green signals gives yellow color, which is indicative of colocalization of the two proteins. (B) As in A except that the cells were stained with a polyclonal anti-hMSH3 antibody (green). (C) As in A except that cells were labeled with BrdU for 1 h before fixation, and the nuclei then were stained with anti-BrdU antibodies (green). (D) As in C except that the nuclei were stained with anti-hMSH3 polyclonal antibody. Note that the MMR protein signals in A and B are green, whereas those in C and D are red.

Complementation of the MMR defect of HCT15 cells in vitro and in vivo. (A) Western blot of cytoplasmic extracts used in MMR assays shown in B. (Lanes 1,2) 50 μg HeLa and HCT15, respectively; (lane 3) HCT15 cell extract (50 μg) supplemented with 20 ng of recombinant hMutSα; (lane 4) HCT15 cell extract (50 μg) supplemented with 20 ng of recombinant hMSH6Δ77/hMSH2; (lane 5) extract of HCT15 cells stably transfected with hMSH6 cDNA (50 μg); (lane 6) extract of HCT15 cells stably transfected with hMSH6Δ77 cDNA (50 μg). (B) Mismatch repair efficiencies of extracts shown in A; lane number correspond with those in A. The light gray column indicates the repair efficiency of HCT15 extract supplemented with 200 ng of recombinant hMSH6Δ77/hMSH2.

Consensus PCNA-binding motif. (A) Alignment of the conserved PCNA-binding motifs of hMSH6, hMSH3, p21^Cip1/WAF1 (hCDN1), flap endonuclease FEN1 (hFEN1), DNA methyltransferase I (hMTDM), XPG endonuclease (hXPG), DNA ligase I (hDNL1), uracil DNA glycosylase (hUNG), and the small subunit of DNA polymerase-δ (hp66). (B) Evolutionary conservation of the putative PCNA-binding motifs of hMSH6 and hMSH3 homologs from human (Homo sapiens), mouse (Mus musculus), budding yeast (S. cerevisiae), fission yeast (Schizosaccharomyces pombe), and Arabidopsis thaliana. The residues important for binding are shown in boldface. Basic residues in the flanking sequences are in gray, and proline residues are underlined. The numbers in brackets denote the amino acid residues of the respective PCNA-binding motifs. (C) Schematic representation of hMSH6, hMSH6Δ77 (which lacks 77 N-terminal amino acid residues of hMSH6), and hMSH3, showing location of the PCNA-binding motifs, the proposed sites of interaction with hMSH2, as well as the positions of the mismatch-, ATP- and magnesium-binding sites. The numbers represent amino acid residues; numbers in brackets above hMSH6Δ77 refer to the corresponding amino acid residues in full-length hMSH6.

Coimmunoprecipitation of hMSH6 or hMSH3 with PCNA. (A) Two hundred micrograms of nuclear extracts of TK6 or HCT15 cells was incubated with the anti-hMSH6 monoclonal antibody 66H6, and the immunoprecipitates were loaded onto a denaturing SDS–polyacrylamide gel adjacent to lanes containing 20 μg of the untreated extracts of the same lines. The proteins then were electrotransferred onto a nitrocellulose membrane, which was hybridized with antibodies against hMSH6 and hMSH2 (top) or PCNA (bottom). PCNA can be seen to coimmunoprecipitate with hMSH6 in TK6 extracts (lane TK6). No PCNA signal was detectable in coimmunoprecipitates from HCT15 extracts (lane HCT15), which lack full-length hMSH6 (lane HCT15 NE). (IgG) Immunoglobulin light chain. (B) Two picomoles of recombinant hMutSα or hMSH6Δ77/hMSH2 was mixed with 6 pmoles of recombinant PCNA, and the immunoprecipitation was performed with the anti-hMSH6 monoclonal antibody as described in Materials and Methods. No PCNA was coimmunoprecipitated from a mixture with hMSH6Δ77/hMSH2 or when hMutSα was omitted (lane PCNA). (IgG) Immunoglobulin heavy chain. Left lane contains the input proteins (1 pmole hMutSα and 3 pmoles PCNA) loaded on the gel directly without immunoprecipitation. (C) Same as B except that hMutSβ was used in place of hMutSα, and the immunoprecipitations were conducted with a polyclonal anti-hMSH3 antiserum (see Materials and Methods). Left lane contains the reference proteins (1 pmole hMutSβ and 3 pmoles PCNA) loaded directly on the gel without immunoprecipitation. The figures are autoradiographs of the membranes, where the proteins were visualized by ECL (see Materials and Methods).

Colocalization of hMSH6 and PCNA to replication foci in HCT15 cells either untransfected (A) or stably transfected with a vector expressing hMSH6 (B) or the truncated variant hMSH6Δ77 (C). Indirect immunofluorescence of cell nuclei stained with antibodies against hMSH6 (green) or PCNA (red). Superimposition of the red and green signals gives yellow color, which is indicative of colocalization of the two proteins. See text for details.

Inhibition of G/T mismatch repair with peptides carrying the consensus PCNA-binding sites of hMSH3 (amino acids 20–39), hMSH6 (amino acids 3–22), and p21^Cip1/WAF1 (amino acids 140–163). Similar peptide sequences from hMSH2 (amino acids 227–249 and 373–392) and hMLH1 (amino acids 148–167) were used as controls (see Materials and Methods).