(A) Domain structure of hMSH6 and sequence alignment of PWWP modules. Upper panel shows the domain structure of hMSH6. PIP, PCNA-interacting protein motif; NLS, nuclear localization signal. Lower panel shows alignment of representative human PWWP domains. The blue dots indicate residues that form an aromatic cage and bind to H3K36me3. Aligned proteins are hMSH6, BRPF1, WHSC1_N, WHSC1_C, ZDWPW1, and HDGF.
(B) Interactions between PWWP domains and the H3K36me3-containing H3 peptide. The crystal structure of BRPF1 PWWP domain bound to the H3K36me3 peptide (PDB: 2X4Y) is shown in the left panel. BRPF1 does not have the Trp residues and instead contains a PSYP sequence (shown in yellow). The structure of the hMSH6 PWWP domain, which has the authentic PWWP motif, was determined by NMR () in the absence of an H3 peptide (PDB: 2GFU). The H3 peptide in the BRPF1 complex is shown with human hMSH6 (right panel) after superimposing the conserved PWWP domains of the two proteins. The aromatic cage (colored blue) encloses the trimethylated Lys. The rotamer conformations of W106 and F133 in hMSH6 likely have to adjust upon binding of the H3K36me3.

(A) GST pull-down assay for interaction of wild type GST-PWWP peptide with native histone octamer (purified from HeLa cells) or recombinant histone octamer. Unless otherwise mentioned, octamer marked with asterisk in this figure was detected by staining with Coomassie blue, and proteins not marked were detected by silver staining or Western blot using antibodies against the indicated components.
(B) GST pull-down assay for interaction between the native histone octamer and recombinant wild type (WT) or mutant (PAAP) PWWP peptide.
(C) hMutSα pull-down assay by biotin-labeled H3 peptides containing various forms of methylation in K36, as indicated. Bound hMutSα was eluted and detected by an hMSH6 antibody. Relative hMutSα binding was determined using the level of hMutSα in the K36me3 peptide-containing reaction as a reference.
(D) Interaction of H3 peptide containing K36me3 with hMutSα with wild type or a mutant PWWP domain, as indicated. The experiment was performed as in (C).
(E) Recombinant histone H3 with a chemically installed analog of tri-methyllysine at position 36 (H3Kc36me3/Kc36me3) forms histone octamers with other histone proteins (i.e., H2A, H2B, and H4) as efficient as recombinant wild type histone H3 (K36).
(F) Co-immunoprecipitation of histone octamer containing H3Kc36me3 and hMutSα with wild type (WT) or mutant (PAAP) PWWP domain. hMSH2 antibody was used for pulldown. The presence of H3Kc36me3 in the pulldown was detected using an H3K36me3-specific antibody.

(A) In vitro MMR assay to determine hMutSα with wild type (WT) or a mutant (PAAP or Δ340) PWWP domain for its ability to restore MMR to hMSH2-deficient NALM6 nuclear extracts as described (). HeLa nuclear extracts (HL) were used as a positive control. Arrows indicate repair products.
(B) Fluorescence imaging of cells expressing EGFP-hMSH6 containing WT or a mutant PWWP domain in hMSH6-deficient DLD-1 cells in S phase.
(C) Number of hMSH6 foci per nucleus in DLD-1 cells transfected with EGFP-hMSH6 containing WT or a mutant PWWP domain. The “n” value indicates the total number of nuclei analyzed in a given case. The error bars represent SD.
(D) Western blot analysis showing the expression of the indicated proteins in DLD-1 cells stably transfected with a scrambled shRNA (control) or a SETD2-specific shRNA.
(E) Fluorescence imaging of SETD2-knockdown or control DLD-1cells expressing wild type EGFP-hMSH6 in S phase.

(A) Western blot analysis showing the expression of the indicated proteins in HeLa cells stably transfected with a scrambled shRNA (control) and two different SETD2-specific shRNAs (SETD2).
(B) hMSH6 and H3K36me3 foci formation and colocalization in control- and shSETD2-HeLa cells in S or G2/M phase.
(C) The number of hMSH6 foci per nucleus in control- and shSETD2-HeLa cells. The “n” value indicates the total number of nuclei analyzed in a given case. The error bars represent SD.
(D) Western blot analysis showing the abundance of hMSH6 and H3K36me3 in G1, S, or G2/M phase in control HeLa cells. S phase was divided into early (E), middle (M), and late (L) sub-phases.
(E) Quantification of hMSH6 or H3K36me3 expression in different cell cycle phases from three independent Western blots. The intensity of each protein in G1 was used as a reference after correction for loading (i.e., the intensity of tubulin). The error bars represent SD.
Also see .

(A) Analysis of PCR product patterns of four microsatellite markers in subclones derived from control- and shSETD2-HeLa cells. The black Δ or asterisk shows the clone exhibiting deletion of a repeat mark or new repeat species, respectively.
(B) HPRT mutability in control- and shSETD2- HeLa and DLD-1 cells. Data are presented as mean +/− SD. Fold-increase in mutation frequency was calculated using the mutation frequency of control HeLa as a reference, and t-test was used to determine the p value.
(C) In vitro MMR assay using nuclear extracts isolated from control- and shSETD2-HeLa cells.
Also see .

(A) Western blot analysis of SETD2 and H3K36me3 in ccRCC cell lines UOK121 and UOK143.
(B) Nuclear localization of H3K36me3 and hMSH6 in ccRCC cell lines UOK121 and UOK143 in S phase.
(C) The number of hMSH6 foci per nucleus in UOK121 and UOK143 cells. The “n” value indicates the total number of nuclei analyzed in each case. The error bars represent SD.
(D) PCR analysis of microsatellites BAT25, BAT26, and D5S346 in subclones of UOK121 and UOK143 cells. The red Δ or asterisk shows the clone exhibiting deletion of a repeat mark or new repeat species, respectively.
(E) Nuclear localization of H3K36me3 and hMSH6 in UOK143 cells transfected with or without yeast Set2 in S phase.
Also see .

In the late G1 and/or early S phase, SETD2 converts H3K36me2 to H3K36me3. H3K36me3 recruits hMutSα onto chromatin via its interaction with the hMSH6 PWWP domain before DNA replication initiates. During DNA replication, Nucleosome disassembly exposes naked DNA to the replication machinery and also disrupts the H3K36me3-PWWP interaction, which releases hMutSα from histone octamers. hMutSα, which has a strong DNA binding activity, can readily bind to nascent DNA with or without an interaction with PCNA. Once a mismatch is introduced, hMutSα can quickly identify the mismatch to initiate the MMR reaction. Also see .