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First published online 21 November 2006
doi: 10.1242/jcs.03291

Journal of Cell Science 119, 5137-5146 (2006)
Published by The Company of Biologists 2006
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Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest

Marit Otterlei1,2,*, Per Bruheim1,3,§, Byungchan Ahn1,4,§, Wendy Bussen5, Parimal Karmakar1,6, Kathy Baynton7 and Vilhelm A. Bohr1

1 Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224, USA
2 Department of Cancer Research and Molecular Medicine, Laboratory Centre, Faculty of Medicine, Norwegian University of Science and Technology, Erling Skjalgsons gt. 1, N-7006 Trondheim, Norway
3 Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway
4 Department of Life Sciences, University of Ulsan, Ulsan 680-749, Korea
5 Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar St, SHM-C130, New Haven, CT 06515, USA
6 Department of Life Science and Biotechnology, Jadavpur University, Kolkata-700 032, WB, India
7 Research Center, Ste. Justine's Hospital, 3175 Cote Ste. Catherine, Montreal, Quebec H3T 1C5, Canada


Figure 1
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  		Fig. 1. Subcellular localization of WRN-, RAD51-, RAD54-, RAD54B- and PCNA-ECFP and EYFP constructs in live cycling HeLa cells. Cells co-transfected with: pictures 1-3: ECFP-RAD51 and EYFP-WRN. Inset picture 1: a stable ECFP-RAD51 expressing cell after 5 weeks in cell culture; pictures 4-6: ECFP-RAD51 and EYFP-PCNA; pictures 7-9: ECFP-RAD54B and EYFP-WRN; pictures 10-12: ECFP-PCNA and EYFP-RAD54B; pictures 13-15: ECFP-RAD54 and EYFP-WRN; pictures 16-18: ECFP-PCNA and EYFP-RAD54; pictures 19-21: ECFP-RAD51 and EYFP-RAD54; pictures 22-24: ECFP-RAD51 and EYFP-RAD54B.

 

Figure 2
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  		Fig. 2. (A) Survival and growth of HeLa cells treated with MMC. Data from untreated cells and cells exposed to MMC (0.5 and 0.2 µg ml-1) for 16 hours are shown. Each point represents six parallel wells. Error bars are undetectable as they are smaller than the symbols. (B) Co-localization of WRN with RAD51, RAD54B and RAD54 after treatment with MMC. Co-localization of ECFP-RAD51 and EYFP-WRN (upper row), ECFP-RAD54B and EYFP-WRN (middle row), ECFP-RAD54 and EYFP-WRN (lower row) in HeLa cells treated with MMC (0.5 µg ml-1) over night. (C) Re-localization of WRN, RAD54 and RAD54B to PCNA foci after MMC treatment. HeLa (pictures 1-16) and U2OS cells (pictures 17-20) were treated with MMC (0.5 µg ml-1) over night to arrest the cells in S-phase. HeLa cells co-transfected with ECFP-RAD54B, EYFP-WRN, HcRed-PCNA: untreated (pictures 1-4) and treated with MMC (pictures 5-8). HeLa cells co-transfected with ECFP-RAD54, EYFP-WRN, HcRed-PCNA: untreated (pictures 9-12) and treated with MMC (pictures 13-16). Stable EYFP-WRN expressing U2OS cells co-transfected with ECFP-RAD54B and HcRed-PCNA and treated with MMC (pictures 17-20). (D) FRETN values between WRN and the RAD52 epistasis proteins after MMC treatment over night, and between RAD51-RAD54 and RAD51-RAD54B. aRepresents previously published data (Baynton et al., 2003Go) that was taken using the same conditions and settings as the current experiments. It is presented here for comparative purposes. Uuntreated cells. FRETN [FRETN=FRET/(I1 x I3)]1/2 represents FRET normalized against protein expression levels measured from intensities (I) (given as arbitrary units below 250). FRET is calculated from the mean of intensities within a region of interest containing more than 25 pixels. Within the region of interest, all pixels had intensities below 250, and the I levels were between 85-190 for the donor (ECFP) and between 55-155 (EYFP) for the acceptor, respectively. Corresponding pictures from which the data were derived are shown in Fig. 2B and Fig. 1, pictures 19-24 (RAD54-RAD51U and RAD54B-RAD51U). The background level outside foci, i.e. the co-localizing area, is 0, which is also the FRET value determined for more than 95% of foci with co-localizing UNG2-ECFP and UNG2-EYFP proteins.

 

Figure 3
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  		Fig. 3. Co-immunoprecipitation of WRN, RAD51 and ATR. (A) WRN is pulled down with RAD51 (H-92) from whole cell extracts prepared from HeLa cells treated with MMC (0.5 µg ml-1), lane 5. In a WRN pulled down with the rabbit polyclonal anti-WRN (ab17987) we co-immunoprecipitated RAD51, lanes 7 and 8. Mouse anti-WRN (BD) or polyclonal anti-WRN (ab17987) and anti-RAD51 (ab1837) were used for the WBs. (B) WRN is pulled down with ATR from whole cell extracts prepared from HeLa cells. Rabbit polyclonal anti-ATR (ab91) and anti-WRN (ab200) were used for IP, and monoclonal mouse anti-WRN (BD) and anti-ATR (ab91) were use for WB. Input represents whole cell lysate. IgG is normal rabbit IgG (SC).

 

Figure 4
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  		Fig. 4. WRN binds directly to RAD51 and RAD54B. (A) Reciprocal ELISAs showing direct binding between RAD51 and WRN. WRN, RAD52 (positive control), BSA (negative) and RAD51 were coated on microtiter plates. Binding of WRN, RAD52 and RAD51 to each other were measured as described in Materials and Methods. Values (OD490nm) for 1:1 molar ratio between the proteins are given. (B) Relative binding of RAD54B, RAD54, RAD51 and RAD52 to WRN coat in ELISA. Both results (A and B) are representative of one out of at least three independent experiments. Absorbance (OD490nm) is given as mean of duplicates adjusted for background binding to BSA in parallel duplicate wells. (C) Dot blot assay demonstrating a direct interaction between WRN and RAD51, and WRN and RAD54B. PVDF membranes with immobilized proteins were incubated in milk-solutions with or without WRN, and analyzed for WRN binding. (D) RAD51 and RAD54B do not modulate WRN helicase activity on a 12 bp bubble substrate. Native gels showing WRN (6.0 nM and 4.0 nM) mediated unwinding of a 12 bp 5'-labeled bubble substrate after adding increasing amounts of RAD51 (6, 12, 24 and 48 nM) and RAD54B (2, 4, 20 and 40 nM).

 

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© The Company of Biologists Ltd 2006