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Comparative Study
. 2009 Aug 11;48(31):7473-81.
doi: 10.1021/bi900694p.

Physical interaction between replication protein A (RPA) and MRN: involvement of RPA2 phosphorylation and the N-terminus of RPA1

Greg G Oakley  1 , Kristin Tillison, Stephen A Opiyo, Jason G Glanzer, Jeffrey M Horn, Steve M Patrick
Affiliations
Comparative Study

Physical interaction between replication protein A (RPA) and MRN: involvement of RPA2 phosphorylation and the N-terminus of RPA1

Greg G Oakley et al. Biochemistry. .

Abstract

Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2, and RPA3 subunits that binds to single-stranded DNA (ssDNA) with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11-RAD50-NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-double-stranded DNA (dsDNA) junctions or breaks, and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic weakens the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates interactions of RPA with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 shows that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage.

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Figures

Fig. 1
Fig. 1
Diagram of RPA and RPA mutants used in this study. A, Organization of the RPA heterotrimer. B. Amino acid sequences of the RPA2 N-terminal domain used in this study, including wildtype RPA2 (wt-RPA2), alanine and aspartic acid substituted RPA2 (Ala-RPA2, Asp8-RPA2), as well as the predicted residues affected by hyperphosphorylation (Hyp-RPA2). C, RPA constructs containing either truncated (ΔN-RPA1) or omitted (RPA2/3) RPA1.
Fig. 2
Fig. 2
Phosphorylation of RPA2 restricts interactions between NBS1 and RPA. A, Protein expression levels in UMSCC38 cells stably expressing HA-tagged wt-RPA2 and Ala-RPA2, and shRNA-RPA2 constructs were analyzed by separating cell lysates by SDS-PAGE and immunoblotting with a general anti-RPA2 antibody. B, C and E, UMSCC38 cells stably expressing wt-RPA2 or Ala-RPA2 with downregulation of endogenous RPA2 were not treated (0 h) or treated with etoposide (20 μM for 2 h) followed by 4 h and 22 h recovery. B, Cell lysates were separated by SDS-PAGE and immunoblotted with RPA2 antibodies. C, Fixed cells were stained with anti-HA and anti-NBS1 antibodies. D, Cells were scored for RPA-MRN co-localization to DNA damage foci. Two hundred cells were scored per experiment. Error bars indicate standard errors (n = 3). P values were calculated using an unpaired, two-tailed t test. E, Fixed cells were stained with anti-γ-H2AX antibodies.
Fig. 3
Fig. 3
RPA interaction with the MRN complex requires the N-terminus of RPA1 and is regulated by RPA2 phosphorylation. A, Various purified RPAs were used to assess the protein-protein interactions. RPAs (2.5 pmol) were incubated with ssDNA (5 pmol) prior to the addition of the MRN (5 pmol) complex expressed and purified from baculovirus preparations. RPA2 antibodies were used to immunoprecipitate wt-RPA and the RPA mutants. The MRN and RPA2 levels were quantitated with NIH image software. The numbers are the ratios of MRN to RPA2 normalized to the ratio for the ΔNRPA1 sample. RPA, wt RPA; ΔN-RPA1, N-terminal deletion of RPA1 (1-168); RPA2/RPA3, RPA without RPA1 and Asp8-RPA2, a phosphomimetic of RPA2. B and C, Biotin-labeled ssDNA bound to streptavidin coated 96 well plates was incubated with wild-type and mutant RPAs. B, Wt, Asp8-RPA2 and ΔN-RPA1 were added to wells coated with biotin-labeled ssDNA bound to streptavidin. The wells were washed to remove unbound RPA and blocked with BSA and biotin. Purified MRN (5 pmol) was subsequently added to the wells and protein interactions were detected using MRE11 and HRP-conjugated antibodies. The SSB control represents the E. coli single-strand binding protein coating the biotin-labeled DNA prior to MRN addition and the MRN-ssDNA sample represents MRN binding without prior RPA addition. C, The amount of purified RPA (2.5-3.0 pmol) bound to ssDNA was detected with RPA2 and HRP-conjugated antibodies and normalized based on absorbance at 610 nm.
Fig. 4
Fig. 4
RPA binds to MRE11/NBS1 and to NBS1. A, Purified RPA and hyperphosphorylated RPA (HypRPA) (2.5 pmol) were incubated with MRE11/NBS1 (5 pmol) followed by addition of RPA2 antibodies and protein G-agarose beads. Proteins bound to the beads were eluted and separated by SDS-PAGE, and immunoblotted with RPA1, NBS1 and MRE11 antibodies. The numbers are the ratios of MN to RPA1 and normalized to the ratio for the HypRPA sample. B, His-NBS1 (5 pmol) was incubated with RPA (2.5 pmol), and then pulled down with Ni2+ matrix. Proteins bound to the beads were eluted and separated by SDS-PAGE, and immunoblotted with RPA1 and NBS1 antibodies. C, Purified RPA (2.5 pmol) and HypRPA (2.5 pmol) were incubated with purified NBS1 (5 pmol). Immunoprecipitated proteins were separated by SDS-PAGE followed by immunoblotting with NBS1 and RPA1 antibodies. The numbers are the ratios of NBS1 to RPA1 and normalized to the ratio for the HypRPA sample.
Fig. 5
Fig. 5
RPA interacts with both MRE11 and NBS1. A and B, a modified ELISA was performed as described under Material and Methods. Purified RPA, Asp8-RPA2, ΔN-RPA1 and RPA2/3 (2.5 pmol) were pre-incubated with ssDNA (2.5 pmol) before addition to the 96 well plates coated with purified (A) NBS1 or (B) MRE11 (5 pmol/well). Primary antibodies directed against RPA2 were added followed by the addition of HRP-conjugated secondary antibodies. The absorbance at 610 nm was measured over a period of 20 min. The relative absorbance values represent the affinity of the RPA forms for NBS1 and MRE11.
Fig. 6
Fig. 6
The basic cleft of the N-terminal domain of RPA1 is required for RPA-MRN interaction. Cells stably expressing V5-RPA1 or V5-A31A41-RPA1 were not treated or treated with etoposide (50 μM for 2 h). Chromatin lysates were prepared and incubated with anti-V5 agarose beads. In A, the immunoprecipitated proteins were separated by SDS-PAGE, followed by immunoblotting with antibodies to NBS1 and RPA1. B, Cells are treated with RPA1-siRNA or non-targeting siRNA (NT) for 48 h. Cell lysates were separated by SDS-PAGE and immunoblotted with RPA1 antibodies. C, UMSCC38 cells and cells expressing V5-wt - and V5-Ala31/Ala41-RPA1 were treated with RPA1-siRNA or non-targeting siRNA (NT) for 48 h. Cells were then exposed to 50 μM etoposide for 2 h or not treated (0 h) followed by 10, 16, and 22 h recovery. DNA content was measured by flow cytometry. The results are representative of two independent experiments. In D, the quantification of the percent of cells in G2 is presented.
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