QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 2 September 2008
doi: 10.1242/jcs.026393

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hong, Z. Articles by Yasui, A. Search for Related Content PubMed PubMed Citation Articles by Hong, Z. Articles by Yasui, A. Social Bookmarking                         What's this?

Recruitment of mismatch repair proteins to the site of DNA damage in human cells

Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Seiryomachi 4-1, Aobaku, Sendai 980-8575, Japan

View larger version (62K): [in this window] [in a new window]   Fig. 1. Accumulation of MSH2, MSH3 and MSH6 at the sites of DNA strand breaks. Accumulation of MSH2, MSH3 and MSH6 at the laser-irradiated site. Immediately after laser irradiation cells were stained with antibodies against MSH2, MSH3 and MSH6 in HeLa cells (A), MSH3 and MSH6 in HCT116 cells (B, upper two lines of panels) and MSH2 in LoVo cells (B, bottom panels). Scanned site is indicated with a white arrow. H2AX was stained and the images were merged. (C) Accumulation of MMR proteins at UVDE-induced SSBs and their colocalization with XRCC1 in XPA-UVDE cells after local UV irradiation (20 J/m2). (D) Influence of the poly(ADP-ribose) polymerase inhibitor DIQ on the accumulation of MSH2 after UV irradiation in XPA-UVDE cells (upper panels) and after laser irradiation in HeLa cells (lower panels).

View larger version (52K): [in this window] [in a new window]   Fig. 2. Accumulation of GFP-tagged MSH2, MSH3 and MSH6 at the laser-irradiated site. (A) Accumulation kinetics of GFP-tagged MSH2, MSH3 and MSH6 at laser-irradiated site in HeLa cells. (B) GFP-MSH3 and MSH6 but not mDR-MSH2 accumulate at laser-irradiated site in HCT116. (C) mDR-MSH2 cotransfected with GFP-MSH3 or with GFP-MSH6 (D) accumulates at laser-irradiated site in HCT116 cells. (E) Western blot analysis of MSH2 in HeLa and LoVo cells (left). Accumulation of GFP-MSH3 and GFP-MSH6 at laser-irradiated site in LoVo cells (right). (F) Accumulation of GFP-MSH3 and GFP-MSH6 at laser-irradiated site after suppression of MSH2 expression in HeLa cells. (G) Influence of suppression of either MSXH3 or MSH6 on the accumulation of GFP-MSH2 at laser-irradiated site in HeLa cells. (H) Influence of suppression of both MSXH3 and MSH6 on the accumulation of GFP-MSH2 at laser-irradiated site in HeLa cells. More than 10 cells are analyzed for each condition and representative data are shown.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Recruitment of MSH3 to laser-irradiated site is mediated by PCNA. (A) Schematic representation of MSH3 and its deletion mutants and the results of recruitment experiments. Black and gray boxes indicate PCNA-binding motif and MSH2-binding domains, respectively. The site of mutation (F27A/F28A) is indicated with a cross. (B) GFP-MSH3 colocalizes with endogenous PCNA at sites of laser-irradiation. GFP-PCNA is recruited to the site of laser-irradiation in HCT116 cells (right panel). (C) Coexpression of GFP-MSH3 with mDR-PCNA wild-type (upper panels) or with mDR-PCNA mutant (D41A) (lower panels) in HeLa cells. (D) mDR-MSH2 was not recruited to the site of laser-irradiation by coexpression of the GFP-tagged mutant MSH3 (F27A/F28A) in HCT116 cells. Arrows indicate the sites of irradiation.

View larger version (39K): [in this window] [in a new window]   Fig. 4. MSH6 is recruited to the site of laser irradiation via its N-terminal region. (A) Schematic representation of the MSH6 and deletion mutants (left) and the results of recruitment experiments (right). Black and gray boxes indicate PCNA-binding motif and MSH2-binding domains, respectively. (B) Recruitment of MSH6 deletion mutants. Arrows indicate the sites of irradiation. (C) Accumulation kinetics of GFP-tagged MSH6 and its deletions. Data are means ± s.e.m. of 3-5 independent experiments. (D) Accumulation of mDR-MSH2 in HCT116 by coexpression of GFP-MSH6 (78-1360).

View larger version (54K): [in this window] [in a new window]   Fig. 5. MLH1 is recruited to the site of laser irradiation via its MutS-binding domain. (A) Colocalization of endogenous MLH1 with H2AX after laser irradiation in HeLa cells (upper panels). There was no accumulation of MLH1 at laser-irradiated site in HCT116 cells (lower panels). (B) Recruitment of GFP-MLH1 to the site of laser irradiation in HeLa cells. (C) Schematic representation of the recruitment of MLH1 deletion mutants. Black and gray boxes indicate PCNA-binding motif and MSH2-binding domain, respectively. The MLH1 deletion mutant containing residues 1-389 is recruited to irradiation sites, whereas that containing residues 389-756, is not.

View larger version (59K): [in this window] [in a new window]   Fig. 6. MSH2 and MSH3 are recruited to UV-induced DNA damage in an NER-dependent manner. (A) Immunostaining of MSH2 and PCNA in locally UV-irradiated (100 J/m2) NER proficient (XPA-WT, labelled WT) and deficient cells (XPA-C2, labelled XPA). (B) Immunostaining of MSH3 and PCNA in locally UV-irradiated (100 J/m2) NER proficient (upper panels) and deficient cells (lower panels). (C) MSH3 is recruited to UV-induced DNA damage via its PCNA-binding motif in XPA-WT cells. GFP-MSH3 (1-57) harboring PCNA-binding domain accumulates at UV-irradiated sites (upper panels), whereas mutation in the PCNA-binding motif and MSH3 without the PCNA-binding domain abrogates the accumulation (middle and bottom panels, respectively).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter