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

First published online 1 September 2005
doi: 10.1242/jcs.02544

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 Lan, L. Articles by Yasui, A. Search for Related Content PubMed PubMed Citation Articles by Lan, L. Articles by Yasui, A. Social Bookmarking                         What's this?

Accumulation of Werner protein at DNA double-strand breaks in human cells

¹ Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Seiryomachi 4-1, Sendai 980-8575, Japan
² Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan
³ Experimental Immunology Branch, NIH, National Cancer Institute, Bethesda, MD 20892-1360, USA
⁴ GeneCare Research Institute, 200 Kajiwara, Kamakura, Kanagawa 247-0063, Japan
⁵ Department of Pathology, University of Washington, Seattle, WA 98195-7470, USA

View larger version (70K): [in a new window]   Fig. 1. Laser irradiation systems and accumulation of WRN. (A) Laser irradiation systems. The left column shows the 365 nm pulse laser irradiation system, producing the lower dose and the higher dose irradiation, which are regulated by the filter in front of the mirror. The right column shows the 405 nm laser system. (B) Three types of damage induced by 365 nm and 405 nm laser irradiation. HeLa cells irradiated with the lower dose or the higher dose of 365 nm laser, or with 405 nm laser of different scan times, were stained with anti-poly(ADP)ribose (left, for single-strand breaks) or by H2AX antibody (middle, for double-strand breaks), respectively, or accumulation of GFP-tagged OGG1 at irradiated sites (right, for base damage) is shown. The amounts of accumulated molecules for poly(ADP)ribose, H2AX and GFP-OGG1 after lower and higher dose irradiation with the 365 nm or 405 nm laser were quantified in the graphs. (C) Time-dependent accumulation of GFP-tagged WRN after higher dose irradiation with the 365 nm laser in HeLa cells. (D) Accumulation of GFP-tagged XRCC1 and LIGIII (upper panels) and no accumulation of GFP-tagged WRN (lower panels) after lower dose irradiation with the 365 nm laser in HeLa cells. Arrows indicate the sites of irradiation.

View larger version (69K): [in a new window]   Fig. 2. Enhanced accumulation of WRN by pre-treatment of cells with BrdU. (A) Accumulation of GFP-tagged WRN indicated by yellow arrows after higher dose irradiation with the 365 nm laser in HeLa cells with or without pre-treatment with BrdU. (B) Accumulation of GFP-tagged WRN after 10 and 100 scans with the 405 nm laser with or without pre-treatment with BrdU in HeLa cells. (C) Immunochemical detection of H2AX after laser irradiation in HeLa cells with or without pre-treatment with BrdU. The number of scans with the 405 nm laser are indicated in yellow. (D) Colocalization of GFP-tagged WRN and H2AX at irradiated sites in HeLa cells after 100 scans with the 405 nm laser. (E) Immunochemical detection of endogenous WRN after 405 nm laser irradiation with or without pre-treatment with BrdU in HeLa cells. The number of scans with the 405 nm laser are indicated in yellow. Arrows indicate the sites of irradiation.

View larger version (24K): [in a new window]   Fig. 3. Comparison of accumulation kinetics of GFP-tagged WRN, NBS1, BRCA1 and LigIII after laser irradiation of HeLa cells. HeLa cells transfected with the various GFP-tagged genes were irradiated with high-dose laser irradiation, and accumulation and dissociation kinetics of the proteins were quantified.

View larger version (26K): [in a new window]   Fig. 4. Influence of RO-19-8022 on accumulation of GFP-WRN in HeLa cells. After higher dose irradiation with the 365 nm laser in HeLa cells, accumulation of WRN was not affected (A), whereas that of GFP-tagged NTH1 was affected (B) by pre-treatment with RO-19-8022.

View larger version (80K): [in a new window]   Fig. 5. Replication-independent accumulation of WRN at irradiated site. Accumulation of GFP-tagged WRN in G1/S, S and G1 phase HeLa cells after higher dose irradiation with the 365 nm laser is shown. At least ten cells from each cell-cycle phases were irradiated, and representative data are shown. Arrows indicate the sites of irradiation.

View larger version (25K): [in a new window]   Fig. 6. WRN accumulates at double-strand breaks via its HRDC domain in vivo. (A) Domains and mutations introduced in GFP-WRN. (B) Results of accumulation of the mutants of GFP-WRN at double-strand breaks. + means positive and – means negative accumulation. (C) Accumulation kinetics of GFP-tagged full-length WRN (WRN) and the HRDC domain (HRDC) after laser irradiation of HeLa cells. Standard deviations derived from at least three independent data are indicated.

View larger version (93K): [in a new window]   Fig. 7. Accumulation of WRN after higher dose laser irradiation at double-strand breaks in the cell lines defective in various putative WRN-interacting proteins. Accumulation of GFP-tagged WRN indicated by yellow arrows 3 minutes after higher dose irradiation with the 365 nm laser in corresponding cell lines. Arrows indicate the sites of irradiation.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter