The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress

Abstract

Proteins with annealing activity are newly identified ATP-dependent motors that can rewind RPA-coated complementary single-stranded DNA bubbles. AH2 (annealing helicase 2, also named as ZRANB3) is the second protein with annealing activity, the function of which is still unknown. Here, we report that AH2 is recruited to stalled replication forks and that cells depleted of AH2 are hypersensitive to replication stresses. Furthermore, AH2 binds to PCNA, which is crucial for its function at stalled replication forks. Interestingly, we identified a HARP-like (HPL) domain in AH2 that is indispensible for its annealing activity in vitro and its function in vivo. Moreover, searching of HPL domain in SNF2 family of proteins led to the identification of SMARCA1 and RAD54L, both of which possess annealing activity. Thus, this study not only demonstrates the in vivo functions of AH2, but also reveals a common feature of this new subfamily of proteins with annealing activity.

Figure 1. AH2 is involved in cellular response to DNA damage

(A) HeLa cells were laser-microirradiated and analyzed by immunostaining with AH2 and RPA2 antibodies. (B) HeLa cells were transfected with the plasmid encoding SFB-tagged AH2. Immunostaining experiments were performed 6 hr after HU treatment using indicated antibodies. Foci-positive transfected cells were quantified by counting a total of 100 transfected cells with positive staining. Data are presented as mean ± s.d. from 3 different experiments. (C) Knock-down efficiency of AH2 using specific shRNAs was confirmed by immunoblotting of lysates prepared from HeLa cells expressing the indicated shRNA. (D–G) Survival curves in response to increasing doses of HU (D), CPT (E), MMC (F) and IR (G) for indicated cell lines are presented. Cell survival assays were performed as described in the Experimental Procedures. Data are presented as mean ± s.d. from three different experiments. See also Figure S1.

Figure 2. AH2 is a PCNA-binding protein

(A) Tandem affinity purification was performed using 293T cells stably expressing tagged AH2. The results from mass spectrometry analysis are shown in the table. (B) Association of endogenous AH2 with PCNA in 293T cells was analyzed by immunoprecipitation using anti-AH2 antibody and immunoblotting using antibodies as indicated. (C) AH2 specifically interacts with PCNA. 293T cells were transfected with plasmids encoding SFB-tagged AH2 or other SNF2 family proteins: SMARCD1, SMARCA1, HELLS, RAD54L or HARP. Co-precipitation was carried out using S-protein beads and immunoblotting was performed using antibodies as indicated.

Figure 3. AH2 interacts with PCNA via a conserved PIP box

(A) AH2 contains a PIP box that is highly conserved among some other PCNA interacting proteins. The consensus sequence of PIP box is indicated. (B) The PIP box of AH2 is required for AH2 to bind to PCNA. 293T cells were transfected with plasmids encoding SFB-tagged wild-type AH2 or three PIP box mutants (dPIPB, Q519A and F525A) of AH2. Co-precipitation was carried out using S-protein beads and immunoblotting was performed using antibodies as indicated. (C) AH2-depleted (AH2 shRNA#55) HeLa derivative cell lines stably expressing shRNA-resistant wild-type AH2 or three PIP box mutants (dPIPB, Q519A and F525A) of AH2 were generated. The empty vector was included as control. The endogenous and exogenous AH2 expression was confirmed by immunoblotting using indicated antibodies. (D) PIP box mutants of AH2 could not rescue HU hypersensitivity of AH2-depleted cells. Survival curves are shown for indicated cell lines in response to increasing doses of HU. Data are presented as mean ± s.d. from three different experiments. See also Figure S2.

Figure 4. A C-terminal conserved region in AH2 is required for its tethering to DNA damage sites

(A) HeLa cells were transfected with the plasmid encoding SFB-tagged AH2 and dPIPB mutant. Immunostaining experiments were performed 6 hr after HU treatment using indicated antibodies. (B) Schematic representation of wild type and mutant AH2 used in this study. Four distinct conserved regions are presented: SNF2 helicase domain (residues 45–498); PIP box (residues 510–528); HARP-like (HPL) domain (residues 712–820); and HNH motif (residues 1011–1051). (C and D) Experiments were performed as in (A). (E) Foci-positive transfected cells were quantified by counting a total of 100 transfected cells with positive staining. Data are presented as mean ± s.d. from 3 different experiments. (F) Deletion of C-terminal conserved region (dC80) failed to rescue HU hypersensitivity in cells with AH2 depletion. AH2 depleted (AH2 shRNA#55) HeLa derivative cell lines stably expressing shRNA-resistant wild-type AH2 or dC80 mutant were generated. The empty vector was included as control. Survival curves are shown for indicated cell lines in response to increasing doses of HU. Data are presented as mean ± s.d. from three different experiments. (G) The endogenous and exogenous AH2 expression was confirmed by immunoblotting using indicated antibodies. See also Figure S2 and S3.

Figure 5. HARP-like domain dictates the annealing activity and the in vivo function of AH2

(A) Schematic diagram of the annealing activity assay. Plasmid DNA is incubated with purified RPA and topoisomease I to generate partially unwound DNA molecules with stably-bound RPA. Addition of SDS and proteinase K to the partially unwound DNA to inactivate and deproteinize topoisomerase I will result in the generation of negatively supercoiled DNA due to unwinding of the DNA by RPA. However, if any protein with annealing activity rewinds the partially unwound DNA in the presence of topoisomerase I, the resulting DNA would be relaxed prior to SDS treatment and deproteinization. (B) HARP-like (HPL) domain dictates the annealing activity of AH2. The annealing helicase assay was carried out as described in the Experimental Procedures, with 60 nM HARP, AH2, AH2N or dHPL in the presence (top panel) or absence (bottom panel) of ATP. Uridine triphosphate (UTP) was used as control in the absence of ATP. All reactions contained DNA, RPA, and topoisomerase I. (C) ATPase activities of both wild-type and mutant AH2 were stimulated by fork DNA. Graph shows percentage of ATP hydrolyzed against concentration of fork DNA, with mean ± s.d. calculated from three independent experiments. (D) Mutations in the Mg²⁺ binding pocket (AH2-MBM) or ATP binding site (AH2-ABM) of AH2 abolish AH2 annealing activity. The annealing helicase assay was carried out with 60 nM AH2, AH2-MBM and AH2-ABM in the presence of ATP. All reactions contained DNA, RPA, and topoisomerase I. (E) PIP box and HNH motif of AH2 does not contribute to the annealing activity of AH2. The annealing helicase assay were carried similar to those described in (B), with indicated proteins. (F) Deletion of HPL domain failed to rescue HU hypersensitivity in cells with AH2 depletion. AH2-depleted (AH2 shRNA#55) HeLa derivative cell lines stably expressing shRNA-resistant wild-type AH2 or HPL domain deleted mutant (dHPL) of AH2 were generated. The empty vector was included as control. Survival curves are shown for indicated cell lines in response to increasing doses of HU. Data are presented as mean ± s.d. from three different experiments. (G) The endogenous and exogenous AH2 expression was confirmed by immunoblotting using indicated antibodies. See also Figure S4.

Figure 6. AH2 and HARP coordinate to protect stalled replication forks

(A) Knock-down efficiency of AH2 shRNA and HARP siRNA was confirmed by immunoblotting using lysates prepared from HeLa cells in which the indicated shRNA or siRNA had been introduced. (B) Double knockdown of HARP and AH2 considerably induces RPA and γH2AX foci. HeLa cells were introduced with indicated shRNAs or siRNAs. Seventy-two hours later, cells were subjected to immunostaining using indicated antibodies. The quantification of foci-positive cells was performed by counting a total of 200 cells per sample. Data are presented as mean ± s.d. from 3 different experiments. (C) Double knockdown of HARP and AH2 enhances HU sensitivity in HeLa cells. Cell survival assays were performed as described in the Experimental Procedures. Data are presented as mean ± s.d. from three different experiments. (D) Schematic representation of the labeling protocol for DNA fiber analysis of replication forks. Hela cells were pulse labeled with IdU, treated with 2 mM HU for 6 h, and released into medium containing IdU. DNA fibers were prepared on slides and immunolabeled with antibodies to detect the modified thymidine analogs incorporated into the DNA. Representative images of replication forks are shown. (E) Quantification of the percentage of newly initiated replication at the indicated time point following the removal of HU. The percentage of newly initiated replication sites was determined by dividing the number of CIdU-containing tracts by the total of all tracts (IdU-containing tracts + CIdU-containing tracts + IdU and CIdU containing tracts). (F) Quantification of the percentage of stalled replication forks was determined by counting IdU containing tracts. (G) Quantification of the percentage of newly fired origins after the removal of HU was determined by counting CIdU-containing tracts. For all the above quantifications, 100 DNA fibers were counted in each slide and the experiment was repeated three times. The means and s.d. (bars) of three independent experiments are shown with P< 0.001. See also Figure S4 and S5.

Figure 7. Identification and validation of additional proteins with annealing activity that contain HARP-like domain

(A) Schematic representation of HARP-like domain-containing proteins RAD54L and SMARCA1. (B) The annealing helicase assay was carried out using 60 nM AH2, dHPL, RAD54L, SMARCA1 or BRG1 in the presence (top panel) or absence (bottom panel) of ATP. (C) ATPase activity of AH2, RAD54L, SMARCA1, and BRG1 was assayed as described above. Graph shows percentage of ATP hydrolyzed against concentration of fork DNA, with mean ± s.d. calculated from three independent experiments. (D) Mutations in the Mg²⁺ binding pocket (RAD54L-MBM) or the ATP binding site (RAD54L-ABM) of RAD54L eliminate its annealing activity. The annealing helicase assay was carried out with 60 nM RAD54L, RAD54L-MBM or RAD54L-ABM in the presence of ATP. All reactions contained DNA, RPA, and topoisomerase I. See also Figure S6.