DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage

Abstract

Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.

Figure 1

Purification of in vitro hyperphosphorylated RPA. The purification of hyperphosphorylated RPA was performed using the same chromatographic procedures as for rhRPA. The final step in the purification was performed using a Q-Sepharose column. (A) Protein fractions were collected and visualized on a 13% SDS polyacrylamide gel by Coomassie Blue staining. Purified rhRPA (lane 1) and an aliquot of the material loaded on the Q-Sepharose column (lanes 2) were included as controls. Fractions eluted from the Q-Sepharose column are loaded with equal volume (lanes 3–10). The positions of the p70, p34, and p14 RPA subunits are indicated by arrows. (B) Western blot analysis of RPA fractions using the RPA-p34 monoclonal antibody. Lane 1 is the partially purified rhRPA prior to incubation with HeLa crude extracts in the presence of phosphatase inhibitors. Analysis of the sample after incubation of the crude extracts in the presence of phosphatase inhibitors (lane 2), Affi-gel blue pool (lane 3), hydroxylapatite pool (lane 4), Q-Sepharose pool (lane 5), and CIP-treated Q-Sepharose pool (lane 6) are presented.

Figure 2

Phosphospecific antibodies reveal multiple sites of phosphorylation on RPA p34. In vitro hyperphosphorylated RPA was purified and analyzed directly (odd lanes) or was first treated with CIP (even lanes) by SDS–PAGE and Western blot analysis. (A) Blots were probed with the indicated p34 phosphospecific antibodies. (B) Blots were stripped and reprobed with a monoclonal antibody directed against the p34 subunit which detects all forms of RPA.

Figure 3

Mass spectral analysis reveals that the N-terminus of RPA p34 contains at least four independent phosphorylations. (A) Analysis of fraction 39 from the HPLC separation of the solution digest of in vitro hyperphosphorylated RPA indicated that this fraction contained N-terminal peptide corresponding to amino acids 4–42 of the p34 subunit. This fraction was analyzed directly by MALDI-TOF MS (dotted line) or following on-chip treatment of the sample with CIP (solid line). (B) Analysis of the HPLC fractions from the separation of the in-gel digested hyperphosphorylated RPA p34 subunit indicated that fraction 38 contained the peptide corresponding to the p34 N-terminus. This fraction was analyzed directly by MALDI-TOF MS (dotted line) or following on-chip treatment of the sample with CIP (solid line). (C) In vitro phosphorylation of threonine 98 of RPA p34. Analysis of HPLC fraction 29 indicated the presence of a peptide corresponding to amino acids 94–105 of the p34 subunit. MALDI-TOF MS was performed directly (dotted line) or following CIP treatment (solid line). The m/z values, peptide, and modifications corresponding to each peak are indicated in each panel.

Figure 4

Mass spectral analysis reveals the interdomain region and the C-terminal domain of RPA p70 is phosphorylated in vitro. (A) MALDI-TOF mass spectra of HPLC fraction 43 before (dotted) and after CIP treatment (solid) reveals the presence of a phosphopeptide. The mass of the CIP-resistant peak is consistent with tryptic peptide 112–157 from RPA p70. (B) In a separate experiment an additional phosphorylated peak is observed in fraction 43. Treatment of the fraction with CIP confirms the phosphorylation of peptide 112–163 from RPA p70. (C) Mass spectral analysis reveals the C-terminal domain from RPA p70 contains up to four phosphorylations. A phosphate ladder is observed in the MALDI-TOF mass spectrum of HPLC fraction 45 (dotted line). CIP treatment reduces the spectrum to two peaks (solid line) that are identified as peptide 569–600 and this peptide’s methionine oxidation product. The m/z values, peptide, and modifications corresponding to each peak are indicated in each panel.

Figure 5

One- and two-dimensional SDS–PAGE of HeLa and rhRPA demonstrates the presence of multiple RPA-p34 phosphorylations. HeLa cells were treated as described in Materials and Methods and lysates were prepared and probed for RPA-p34. (A) One-dimensional SDS–PAGE and Western blot analysis of extracts prepared from control (lane 1), UV (lane 2), and HU (lane 3) treated cells. Increasing concentrations of purified in vitro hyperphosphorylated RPA (lanes 4–6) or rhRPA (lanes 7–9) were also included on the gel for reference. (B) HeLa lysates (same lysates as depicted in A) and purified hyperphosphorylated RPA were separted by two-dimensional gel electrophoresis as described in Materials and Methods and probed for RPA p34. (C) Enlarged picture of 2D SDS–PAGE of hypRPA and HeLa UV-treated lysates for comparison. Arrows indicate spots that appear to migrate in a similar pattern.

Figure 6

Phosphospecific antibodies reveal multiple sites of phosphorylation on RPA p34 in response to DNA damage. Extracts prepared from HU-treated cells were analyzed directly (odd lanes) or following CIP treatment (even lanes) by SDS–PAGE and Western blot analysis. (A) Blots were probed with the indicated p34 phosphospecific antibodies. (B) Blots were stripped and reprobed with a monoclonal antibody directed against the p34 subunit of RPA which detects all forms of RPA.

Figure 7

Identification of in vivo sites of RPA phosphorylation in response to HU. RPA was purified from HeLa cells treated with HU as indicated in Materials and Methods. The protein was separated by SDS–PAGE, and the p70 and p34 subunits were excised, and subjected to in-gel trypsin digestion, HPLC fractionation, and MALDI-TOF MS analysis. (A) MALDI-TOF MS analysis of the HPLC fraction corresponding to the N-terminus peptide of RPAp34. The corresponding HPLC fraction was analyzed directly (dotted line) or following on-chip treatment of the sample with CIP (solid line). (B) Analysis of the HPLC fraction containing to the peptide corresponding to amino acids 112–157 from the p70 subunit. This fraction was analyzed directly by MALDI-TOF MS (dotted line) or following on-chip treatment of the sample with CIP (solid line). The m/z values, peptide, and modifications corresponding to each peak are indicated in each panel.