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First published online November 19, 2008
doi: 10.1242/10.1242/jcs.033688

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ATR and Rad17 collaborate in modulating Rad9 localisation at sites of DNA damage

Annette L. Medhurst^1,*, Daniël O. Warmerdam^2,*, Ildem Akerman¹, Edward H. Verwayen², Roland Kanaar^2,3, Veronique A. J. Smits^2,, and Nicholas D. Lakin^1,

¹ Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QD, UK
² Department of Cell Biology and Genetics, Cancer Genome Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands
³ Department of Radiation Oncology, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands

View larger version (44K): [in this window] [in a new window]   Fig. 1. Rad9 accumulates in nuclear foci in response to genotoxic stress. (A) HeLa and Swiss 3T3 cells were left untreated, exposed to UV, or treated with aphidicolin. Rad9 was visualised by immunofluorescence. (B) Western blot analysis of untransfected U2OS cells or different U2OS clones stably expressing GFP-Rad9 (II-5 and II-19), using the indicated antibodies. (C) U2OS cells or clones expressing GFP-Rad9 (II-5 and II-19) were lysed and GFP-Rad9 immunoprecipitated using anti-GFP antibodies. The presence of associated proteins was analysed by immunoblotting with the indicated antibodies. (D) U2OS cells or cells expressing GFP-Rad9 were either left untreated or exposed to UV. After 1 hour, cells were lysed, and western blot analysis was performed using the indicated antibodies. (E) U2OS cells expressing GFP-Rad9 were left untreated, exposed to UV, or treated with aphidicolin. GFP-Rad9 was visualised by direct fluorescence. (F) U2OS cells expressing GFP-Rad9 were left untreated or exposed to UV. GFP-Rad9 was detected by direct fluorescence and RPA (p34 subunit) by immunofluorescence.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Rad17 is required for Rad9 to form foci in response to DNA damage. (A) HeLa cells were transfected with siRNA oligonucleotides as indicated and were left untreated or exposed to aphidicolin. Whole cell extracts were prepared for western blotting (left panel) and the percentage of cells exhibiting more than ten Rad9 foci was determined (right panel). (B) U2OS cells expressing GFP-Rad9 were transfected with empty vector, or vector containing Flag-Rad17 or Flag-Rad17AA. Cells were left untreated or exposed to aphidicolin. Chromatin fractions were prepared and extracts subjected to western blotting using antibodies as indicated. (C) U2OS cells stably expressing GFP-Rad9 were transfected with Flag-Rad17 or Flag-Rad17AA constructs prior to treating cells with aphidicolin. Flag-positive (transfected) or Flag-negative (untransfected) cells were identified by immunofluorescence and cells scored for GFP-Rad9 foci. (D) U2OS cells stably expressing GFP-Rad9 were transfected as in C, after which cells were treated with UV. Flag-positive (transfected) or Flag-negative (untransfected) cells were identified by immunofluorescence and cells were scored for GFP-Rad9 foci.

View larger version (38K): [in this window] [in a new window]   Fig. 3. ATR is required for formation of a subset of UV- and aphidicolin-induced Rad9 foci. (A) HeLa cells were transfected with siRNA oligonucleotides as indicated, exposed to UV and chromatin fractions prepared. Western blot analysis was performed using the indicated antibodies. (B) HeLa cells were transfected with siRNA oligonucleotides as indicated. Cells were treated with UV and harvested for western blotting (upper panel). The percentage of cells exhibiting more than ten Rad9 foci was determined by immunofluorescence (lower panel). *P=0.017 between these two data points indicating statistical significance at the 95% confidence level. (C) Cells were transfected with siRNA and treated as in B. The percentage of cells exhibiting more than ten RPA (p34 subunit) foci was determined by immunofluorescence. (D) HeLa cells were transfected with siRNA oligonucleotides as indicated and 72 hours after transfection were treated with a combination of ATM (10 µM) and DNA-PK (1 µM) inhibitors, or left untreated for 1 hour prior to exposure to aphidicolin. Cells were fractionated to obtain chromatin-enriched proteins and western blotting performed using the indicated antibodies. (E) HeLa cells were transfected with siRNA oligonucleotides as indicated and whole cell extracts prepared for western blotting (upper panel). In parallel, cells were treated with ATM (10 µM) and/or DNA-PK (1 µM) inhibitors, or left untreated prior to exposure to aphidicolin. The percentage of cells exhibiting more than ten Rad9 foci was determined.

View larger version (43K): [in this window] [in a new window]   Fig. 4. ATR influences the retention time of GFP-Rad9 in damage-induced foci. (A) U2OS cells expressing GFP-Rad9 were transfected with ATR or Luciferase siRNA oligonucleotides, and treated with aphidicolin. Half the nucleus containing GFP-Rad9 foci (see rectangle) was bleached for 2.7 seconds at 100% laser intensity, after which redistribution of fluorescence was monitored by recording images every 60 seconds. Western blot analysis of cell extracts from a representative experiment is illustrated (right panel). (B) Quantification of simultaneous FLIP-FRAP experiment as in A, with the exception that the cells were imaged every 30 seconds. Cells were treated with aphidicolin (left panel) or UV (right panel), after which FLIP was measured in the unbleached part of the cell and FRAP was measured in the bleached part of the cell. Control cells (UNT) are either left untreated or treated with damaging agents but not displaying GFP-Rad9 foci. The mean of the data points of individual cells analysed is illustrated (n=number of cells) with error bars representing the s.e.m. (C) Difference in relative fluorescence in bleached and unbleached parts of the nucleus from B, plotted against time. Error bars represent twice the s.e.m.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Rad17 phosphorylation at Ser635 and Ser645 decreases the mobility of GFP-Rad9 in damage-induced foci. U2OS cells expressing GFP-Rad9 were transfected with Flag-Rad17 or Flag-Rad17AA constructs, together with mCherry-C1, to detect transfected cells. Cells were left untreated (UNT) or treated with UV and FLIP-FRAP analysis was performed on red cells. (A) Quantification of FLIP and FRAP as described in Fig. 4B. (B) Difference in relative fluorescence, plotted against time, as described in Fig. 4C. (C) ATR and Rad17 collaborate in modulating Rad9 localisation at sites of DNA damage. ssDNA generated as a result of DNA damage or replication stress is recognised by RPA (1). Recognition of ssDNA by RPA leads to the independent recruitment of ATR and Rad17 to DNA lesions. Rad17 loads the 9-1-1 complex at sites of ssDNA and facilitates activation of ATR through an interaction with TopBP1 (2). ATR subsequently phosphorylates Claspin, which acts to recruit Chk1 and promote its phosphorylation by ATR. ATR also phosphorylates Rad17 (3). ATR-mediated phosphorylation of Rad17 stabilises the 9-1-1 complex at sites of DNA lesions. This could in turn result in the maintenance of activated ATR and continued checkpoint signalling until DNA damage is repaired (4).

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