The annealing helicase HARP is recruited to DNA repair sites via an interaction with RPA

Abstract

HepA-related protein (HARP) (also known as SMARCAL1) is an ATP-driven annealing helicase that catalyzes the formation of dsDNA from complementary Replication protein A (RPA)-bound ssDNA. Here we find that HARP contains a conserved N-terminal motif that is necessary and sufficient for binding to RPA. This RPA-binding motif is not required for annealing helicase activity, but is essential for the recruitment of HARP to sites of laser-induced DNA damage. These findings suggest that the interaction of HARP with RPA increases the concentration of annealing helicase activity in the vicinity of ssDNA regions to facilitate processes such as DNA repair.

Figure 1.

Purification of a HARP-containing complex. Nuclear extracts were prepared from normal HeLa S3 cells (−) or HeLa cells expressing Flag-HA-tagged HARP (+). The proteins were subjected to sequential anti-Flag and anti-HA immunoaffinity purification, and then analyzed by SDS–polyacrylamide gel electrophoresis and silver staining. The polypeptides that consistently copurified with Flag-HA-HARP were identified by mass spectroscopy and are indicated.

Figure 2.

The conserved N-terminal region of HARP is required for the interaction of HARP with RPA. (A) Schematic representation of the conserved regions of HARP. The human, mouse, frog, and chicken HARP proteins were aligned by using the Clustal W algorithm, and conserved amino acid residues with 100% identity are represented by black hatches. The amino acid sequences of the N-terminal region are shown below, and the substitutions in the mutant human HARP protein are indicated. (B) Coimmunoprecipitation of RPA and HARP. HARP was immunoprecipitated with anti-Flag resin from nuclear extracts of cells containing wild-type or mutant Flag-HA-HARP. The amino acid substitutions in the mutant HARP protein are as depicted in A. As a control, normal HeLa cells lacking Flag-HA-HARP were also treated in parallel. Western blot analysis of nuclear extract (NE) and immunoprecipitated samples (IP) was performed by using anti-HARP and anti-RPA2 antibodies.

Figure 3.

The RPA interaction region of HARP is not required for annealing helicase activity. (A) Purification of wild-type and mutant HARP proteins. Recombinant Flag-HARP proteins were synthesized by baculovirus expression in Sf9 cells, purified by Flag affinity chromatography, and analyzed by SDS–polyacrylamide gel electrophoresis and staining with Coomassie Blue R-250. The amino acid substitutions in the mutant HARP protein are depicted in Figure 2A. (B) The conserved N-terminal region of HARP is necessary for the binding of purified HARP to purified RPA. Wild-type or mutant Flag-HARP was incubated with purified recombinant RPA, and then immunoprecipitated with anti-Flag resin. After the beads were washed, the bound proteins were eluted and detected by Western blot analysis. (C) The N-terminal 36-amino-acid residues of HARP are sufficient for binding to RPA. The wild-type and mutant versions of the N-terminal 36-amino-acid residues of HARP (fused to a C-terminal Flag tag) were synthesized in bacteria and purified to near homogeneity. The binding of these peptides to purified RPA was analyzed, as in B. (D) Both wild-type and mutant HARP proteins possess ATP-dependent annealing helicase activity. Annealing helicase assays were carried out as described by Yusufzai and Kadonaga (2008). HARP-mediated DNA rewinding is monitored by the ATP-dependent relaxation of RPA-unwound DNA in the presence of topoisomerase I. An equimolar amount of UTP was used as a control for the absence of ATP.

Figure 4.

Wild-type but not RPA-binding-defective mutant HARP localizes to laser-induced damage sites. (A) Endogenous HARP protein is localized to sites of DNA damage in several different cell lines. The indicated cells were microirradiated with a nanosecond green laser. After 1 h, the cells were fixed and stained with anti-HARP antibodies. The nuclei are outlined with a dotted line. (B) Cells containing wild-type or mutant Flag-HA-HARP were microirradiated, as in A. After 30 min, cells were fixed and stained with anti-HARP and anti-Flag antibodies. The anti-HARP antibodies recognize both the endogenous and transgenic HARP, whereas the anti-Flag antibodies recognize only the transgenic HARP. All of the wild-type HARP-containing cells (N = 8) exhibited specific localization of HARP to the DNA damage sites, whereas none of the mutant HARP-containing cells (N = 8) showed localization of the mutant HARP to the DNA damage sites. (C) A simple view of HARP protein.