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First published online 14 November 2007
doi: 10.1242/jcs.004580

Journal of Cell Science 120, 4221-4229 (2007)
Published by The Company of Biologists 2007
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NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse

Karoline C. Manthey1, Stephen Opiyo2, Jason G. Glanzer1, Diana Dimitrova1, James Elliott3,* and Gregory G. Oakley1,{ddagger}

1 Department of Oral Biology, University of Nebraska Medical Center College of Dentistry and Nebraska Center for Cellular Signaling, Lincoln, NE 68583, USA
2 Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE, USA
3 Department of Environmental Health, University of Cincinnati, Cincinnati, OH, USA


Figure 1
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  		Fig. 1. Anti-NBS1 and anti-NBS1-S(P)343 block the formation of RPA32-S(P)4-S(P)8 and RPA foci. (A-R) Following the introduction of the negative-control IgG (A-F), anti-NBS1 (G-L) or anti-NBS1-S(P)343 (M-R), HeLa cells were treated with 5 mM HU, or were mock-treated (control), for 3 hours. Cells were detergent-extracted and fixed, incubated with primary and secondary antibodies, and visualized by confocal microscopy. (D-F) HU treatment led to RPA32-S(P)4-S(P)8 (green) and RPA32 (red) focus formation in negative-control IgG-transfected cells. (J-L,P-R) anti-NBS1-and anti-NBS1-S(P)343-transfected cells contained RPA32 foci but RPA32-S(P)4-S(P)8 foci were not observed. HU-induced damage was verified by H2AX phosphorylation (supplementary material Fig. S4).

 

Figure 2
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  		Fig. 2. Impaired formation of RPA32-S(P)4-S(P)8 foci in NBS-ILB1 (vector only) and NBS-ILB1 (S343A) cells. (A-R) The indicated isogenic cell lines were treated with 5 mM HU for 3 hours prior to detergent-extraction, fixation and immunofluorescence using RPA32-S(P)4-S(P)8 (green) or RPA32 (red) antibodies. (D-F) HU treatment induced RPA recruitment to sites of DNA damage and normal RPA hyperphosphorylation occurred in NBS-ILB1 (NBS1) cells. (J-L,P-R) NBS-ILB1 (vector only) and NBS-ILB1 (S343A) cells recruited RPA to sites of DNA damage; however, RPA32 was not phosphorylated at these sites. HU toxicities were comparable for all cell lines tested (supplementary material Fig. S3).

 

Figure 3
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  		Fig. 3. NBS1 and ATR are required for RPA phosphorylation following HU-induced replicative stress. (A) NBS-ILB1 (NBS1), NBS-ILB1 (S343A), NBS-ILB1 (S278A/S343A) and NBS-ILB1 (LXIN, vector only) cells were synchronized with 6 µM aphidicolin for 16 hours. The medium was replaced with fresh medium for 3 hours before HU treatment. Following 3 hours of treatment with 2 mM HU, cells were harvested and chromatin fractions were prepared. Proteins were detected by immunoblotting with the corresponding antibodies. (B) Chromatin isolated from the A-T cell line AT22IJE-T (ATM+) and its isogenic derivative cell line expressing recombinant ATM (ATM–) showed little difference in RPA-chromatin binding or RPA phosphorylation. Similar to the A-T cell lines, U2OS cells expressing either wild-type ATR or kinase-dead ATR (ATR-kd) displayed similar RPA-chromatin binding following DNA damage. Distinct from A-T cells, ATR-kd expression in U2OS cells significantly decreased RPA hyperphosphorylation.

 

Figure 4
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  		Fig. 4. HU-induced ATR foci and chromatin retention is decreased both in NBS1-deficient and NBS1 phospho-mutant cells. (A-R) NBS-ILB1 (NBS1), NBS-ILB1 (vector only) and NBS-ILB1 (S278A/S343A) cells were synchronized and treated with 2 mM HU for 3 hours. To visualize detergent-resistant ATR foci, cells were extracted with detergent before fixation. (D-F) In NBS cells restored with wild-type NBS1, ATR foci (green) were still visible after detergent extraction (D) and ATR foci colocalized with HU-induced RPA32 foci (red) (F). (G-R) Decreased retention of ATR (J,P) and a loss of colocalization with RPA (L,R) was observed in NBS1-deficient and in NBS1 phospho-mutant cells. (S) ATR retention in chromatin fractions of synchronized NBS-ILB1 (NBS1) and NBS-ILB1 (S278A/S343A) cells was examined by immunoblotting with anti-ATR antibodies. NBS cells expressing NBS1 retained ATR in the chromatin fractions, whereas NBS cells expressing a phospho-mutant form of NBS1 (S278A/S343A) did not retain ATR in the chromatin. Orc2-chromatin binding was not altered following DNA damage and was used as a loading control.

 

Figure 5
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  		Fig. 5. Wild-type HA-RPA32-containing heterotrimers are hyperphosphorylated following siRNA downregulation of endogenous RPA32 and HU treatment. (A) SCC38 cells were transfected with HA-wt-RPA32 (lanes 2,3) or HA-A4A8-RPA32 (lanes 3,4). Cells were treated with siRNA directed towards the 3' non-coding sequence of RPA32 for 48 hours. Following siRNA downregulation of endogenous RPA32, cells were subjected to HU (5 mM) treatment for 3 hours. Lysates from either control or HU-treated cells were analyzed for the presence of RPA32 by western blot analysis using RPA32 antibodies that recognize both endogenous and transfected RPA32. The membrane was reblotted without stripping with antibodies that recognize phosphorylated S4S8 forms of RPA32 (bottom panel). (B) HA-tagged RPA32 formed a complex with endogenous RPA70 and RPA14, as demonstrated by co-immunoprecipitating HA-tagged RPA32. Lysates prepared from UM-SCC38 cells transfected with an empty vector or HA-wt-RPA32 were subjected to immunoprecipitation using HA antibodies. Immunoprecipitates were then analyzed for RPA70, RPA32 and RPA14 by western blot analysis (lanes 1-2).

 

Figure 6
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  		Fig. 6. Loss of S4S8 phosphorylation suppresses HU-induced apoptosis. (A) Time of onset of apoptosis after 24 hours of HU treatment (2 mM). HU-induced apoptosis was assessed by Annexin V staining and flow cytometry. Right-hand quadrants indicate Annexin-V-positive cells, indicative of apoptosis. Cells in the upper quadrants have begun to lose membrane integrity and stain positive for propidium iodide and Annexin V. The results from one of three representative experiments are shown. (B) The percentage of apoptotic cells was calculated from at least three separate experiments. The percentage is presented as the mean ± s.d. of the total number of gated cells in the upper-and lower-right quadrants. Following siRNA downregulation of endogenous RPA32, cells were treated with 2 mM HU for 24 hours. At the indicated time points, cells were harvested, fixed and stained with Annexin V to detect apoptotic cells.

 

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© The Company of Biologists Ltd 2007