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. 2005 Nov 1;391(Pt 3):473-80.
doi: 10.1042/BJ20050379.

Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage

Xiaoming Wu  1 , Zhengguan Yang, Yiyong Liu, Yue Zou
Affiliations

Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage

Xiaoming Wu et al. Biochem J. .

Abstract

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

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Figures

Figure 1
Figure 1. Induction of RPA hyperphosphorylation by genotoxic agents in human cells
(A) Exponentially growing A549 cells were treated with indicated doses of UV followed by a 2 h recovery. Total cellular lysates were prepared and then subjected to Western blotting with anti-RPA32 and anti-actin antibodies respectively. (B, C) A549 cells were treated with indicated doses of CPT (B) or HU (C) for 3 h. Total cell lysates were prepared for Western blotting with anti-RPA32 and anti-actin antibodies respectively. (D) A549 cells were treated with 10 J/m2 UV, 2 μM CPT and 1 mM HU (lanes 2–4) respectively, as indicated above. Whole cell lysates were prepared and probed with anti-RPA32 (upper panel) and anti-phospho-RPA32 Ser4/Ser8 (lower panel) antibodies respectively. The arrows indicate the hyperphosphorylated RPA.
Figure 2
Figure 2. Co-IP of Rad51 and Rad52 with hyperphosphorylated RPA after DNA damage
(A) Exponentially growing cells were treated with increasing doses of UV irradiation or CPT as above, and then total cell lysates were prepared and probed with anti-γH2AX and anti-actin antibodies respectively. (B) Cells were treated with the indicated doses of UV irradiation followed by a 2 h recovery or treated with the indicated doses of CPT for 3 h. Nuclear extracts were prepared and analysed by Western blotting with anti-Rad52 or anti-Rad51 antibodies. (C, D) Cells were treated with indicated doses of UV or CPT and then total cell lysates were prepared for co-IP assays with anti-Rad51 and anti-Rad52 antibodies. Proteins from the immunoprecipitates were detected by Western blotting using anti-RPA32 antibody. As control, 10% of the total volumes of the whole cell lysates used for the IP were also included (Input, upper panel). The hyperphosphorylated RPA immunoprecipitated with Rad51 and Rad52 was quantified by densitometry and further normalized to the inputs that were designated as of value 1. (E) Total cell lysates prepared from 20 J/m2 UV irradiated cells (lanes 1 and 3), and 4 pmol in vitro phosphorylated RPA (mixed with 1 pmol native RPA as an internal control) (lanes 2 and 4) were probed by Western blotting with anti-RPA32 (lanes 1 and 2) and anti-phospho-RPA32 Ser4/Ser8 (lanes 3 and 4) antibodies respectively. (F) Total cell lysates prepared from untreated cells were incubated with Rad51 or Rad52 antibodies. The immunoprecipitates were washed three times with PBS containing 0.05% Nonidet P-40, and further incubated in a high concentration of salt buffer (15 mM Tris/HCl, pH 7.5, 600 mM NaCl and 0.1% NP-40) for 30 min at 4 °C to remove endogenously bound proteins (lanes 2 and 3). Then the purified RPA containing a 1:7 mix of in vitro phosphorylated/native protein (lane 1) was added and incubated in RPA binding buffer for 4–6 h. The IPs were washed free of unbound proteins before the bound proteins were detected by Western blotting with RPA32 antibody (lanes 4 and 5). Anti-Rad51, anti-Rad52 or protein A/G beads did not bind non-specifically to the purified RPA (lanes 6 and 7).
Figure 3
Figure 3. Co-localization of RPA with Rad52 and ATR after DNA damage
Cells were mock-treated, treated with 20 J/m2 UV irradiation followed by 2 h recovery or treated with 10 μM CPT for 3 h. After extraction of cytoplasmic proteins, cells were fixed and incubated with primary and secondary antibodies and visualized by fluorescence microscopy. (A) Cells were stained with rabbit anti-Rad52 antibody (green, subpanels B and F) and mouse anti-RPA32 antibody (red, subpanels C and G). Subpanels D and H are the merged images of the anti-Rad52 and anti-RPA32 stained cells. Subpanels A and E are the DAPI (4′,6-diamidino-2-phenylindole)-stained nuclei. (B) Cells were stained with mouse anti-Rad52 antibody (red, subpanels B, F and J) and rabbit anti-phospho-RPA32 Ser4/Ser8 antibody (green, subpanels C, G and K). Subpanels D, H and L are the merged images of the anti-Rad52 and anti-phospho-RPA32 Ser4/Ser8 stained cells. (C) Cells were stained with rabbit anti-ATR antibody (green, subpanels B and F) and mouse anti-RPA32 antibody (red, subpanels C and G). Subpanels D and H are the merged images of the anti-ATR and anti-RPA32 stained cells. Subpanels A and E are the DAPI stained nuclei. (D) Cells were stained with rabbit anti-phospho-RPA32 Ser4/Ser8 antibody (green, subpanels B, F and J) and mouse anti-ATR antibody (red, subpanels C, G and K). Subpanels D, H and L are the merged images of the anti-ATR and anti-phospho-RPA32 Ser4/Ser8 stained cells.
Figure 4
Figure 4. Co-IP of ATR with hyperphosphorylated RPA after DNA damage
(A) Cells were treated with indicated doses of UV irradiation, and then whole cell lysates were prepared and probed with anti-phospho-Chk1-317, anti-ATR and anti-β-actin antibodies respectively. (B) and (C) Cells were treated with indicated doses of UV or CPT, and then whole cell lysates were prepared for co-IP assays with ATR antibody. Proteins from the immunoprecipitates were detected by Western blotting using anti-RPA32 antibody. The arrows indicate the hyperphosphorylated RPA. The hyperphosphorylated RPA immunoprecipitated with ATR was quantified by densitometry and normalized to the inputs that were designated as of value 1.
Figure 5
Figure 5. Interactions of Rad51, Rad52 and ATR with hyperphosphorylated RPA after 60 J/m2 UV irradiation of cells
Cells were treated with 60 J/m2 UV irradiation followed by 2 h recovery and total cellular lysates were prepared for co-IP assays with anti-Rad51 (A), anti-Rad52 (B) or anti-ATR (C) antibodies. Proteins from the immunoprecipitates were detected by Western blotting using anti-RPA32 antibody. Arrows indicate the hyperphosphorylated RPA. The hyperphosphorylated RPA immunoprecipitated with ATR was quantified by densitometry and normalized to the inputs that were designated to be of value 1. (D) Whole cell lysates were prepared from 60 J/m2 UV irradiated cells. Before co-IP assays, the lysates were treated with 200 units of CIAP for 1 h at 37 °C in the absence (−/+) or presence (+/+) of 50 mM glycerophosphate (G.P.), or mock treated (–/–). Treated cell lysates were then subjected to co-IP with anti-Rad52 antibody. The bound proteins were then detected by Western blotting using anti-RPA32 antibody.
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