DNA Binding Properties of the Actin-Related Protein Arp8 and Its Role in DNA Repair

<div><p>Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not γ-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair.</p></div

Binding of Arp8 mutants to DNAs.

<p>(A) Positions of mutations introduced in the ATP binding pocket of Arp8. The crystal structure of the ATP binding pocket of Arp8 was obtained from the Protein Database (PDB ID: 4FO0). (B) Binding of Arp8 and Arp8 mutants to supercoiled and nicked circular forms of φX174. Following concentrations of Arp8 (upper panel, lanes 1 to 10), Arp8 S55A T56A (upper panel, lanes 11 to 20), Arp8 E266A (lower panel, lanes 1 to 10), and Arp8 K288A S290A (lower panel, lanes 11 to 20) were used for this experiment: 0 µM (lanes 1, 6, 11 and 16), 0.8 µM (lanes 2, 7, 12 and 17), 1.6 µM (lanes 3, 8, 13 and 18), 3.2 µM (lanes 4, 9, 14 and 19), 4.8 µM (lanes 5, 10, 15 and 20). In lanes 6 to 10 and 16 to 20, 1 mM ATP was added to the reaction mixture. (C) Intensity of the unbound DNA in the absence or presence of ATP for each Arp8 protein (wild-type Arp8 and Arp8 mutants Arp8 S55A T56A, Arp8 E266A, and Arp8 K288A S290A) used in panel B was quantified and then the data was plotted as relative intensity (%) with respect to that of the unbound DNA from the control (no protein added control) lane.</p

Effect of ATP on the binding of Arp8 to DNAs.

<p>Bindings of actin (lanes 2 to 5) and Arp8 (lanes 6 to 13) to dsDNA (A) and ssDNA (B) were examined in the absence (lanes 2 to 5 and 10 to 13) and presence (lanes 6 to 9) of 1 mM ATP, or in the presence of 1 mM ADP (lanes 14 to 17). Various concentrations of Arp8 (lanes 6–17) and actin (lanes 2–5) were used in this experiment. Concentrations of Arp8 used in panel (A) were: 1.2 µM (lanes 2, 6, 10, and 14), 2.4 µM (lanes 3, 7, 11, and 15), 4.8 µM (lanes 4, 8, 12, and 16), and 7.2 µM (lanes 5, 9, 13, and 17). Concentrations of Arp8 used in panel (B) were: 0.4 µM (lanes 2, 6, 10, and 14), 0.8 µM (lanes 3, 7, 11, and 15), 1.6 µM (lanes 4, 8, 12, and 16), and 3.2 µM (lanes 5, 9, 13, and 17). Lane 1 in both A and B: no protein added control. Intensity of the unbound DNA in each panel was quantified and plotted as before (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108354#pone-0108354-g002" target="_blank">Fig. 2</a> legend).</p

Double-stranded DNA binding activity of purified human Arp8 and its mutants.

<p>(A) Purification of human Arp8. Protein fractions from each purification step were analyzed by SDS-PAGE (gel was stained with Coomassie Brilliant Blue). Lane 1, molecular weight markers. Lanes 2 and 3, whole cell lysates before and after induction with IPTG, respectively. Lanes 4 and 5, peak fractions from Ni-NTA agarose and Heparin Sepharose columns, respectively. Lanes 6 and 7, Heparin Sepharose fraction before and after treatment with PreScission protease (removal of His6 tag). Lane 8, peak fraction from MonoQ column. (B) Purified wild-type and deletion mutants (deletants) of Arp8. Lane 1, molecular weight markers. Lanes 2–4, purified Arp8, Arp8 Δ1–38 (N-terminal deletion), and Arp8 Δ403–463 (insertion IV deletion), respectively. (C) dsDNA binding activities of Arp8 and its deletants. Bindings of Arp8 (lanes 2–6), Arp8 Δ1–38 (lanes 8–12), and Arp8 Δ403–463 (lanes 14–18) to linearized φX174 were examined at various protein concentrations: 0 µM (lanes 1, 7, and 13), 0.4 µM (lanes 2, 8, and 14), 0.8 µM (lanes 3, 9, and 15), 1.6 µM (lanes 4, 10, and 16), 3.2 µM (lanes 5, 11, and 17), and 4.8 µM (lanes 6, 12, and 18). (D)Intensity of the unbound DNA in each lane of panel C was quantified and then plotted as relative intensity (%) with respect to that of the unbound DNA from the control (no protein added control) lane.</p

Characterization of Arp8-knockout cells.

<p>(A) Whole-cell extracts were prepared from same number of wild-type (WT) and <i>ARP8</i>^{-/-/transgene} cells at the indicated times after the addition of 2 µg/ml of tet, and subsequently they were analyzed by Western blot using an anti-Arp8 antibody. (B) Representative growth curves for the WT and <i>ARP8</i>^{-/-/transgene} cells with (tet+) or without (tet−) tetracycline treatment. Results shown are using cells from day 7 to day 13 after the addition of tetracycline. The number of living cells was counted after trypan blue staining and represented as fold increase in cell number. (C) Sensitivity of WT, Arp8 OE and Arp8-knockout (Arp8 KO; <i>ARP8</i>^{-/-/transgene} cells cultured in the presence of tet for 8 days) cells to aphidicolin. Cells were cultured in the absence or presence of 0.25 µM aphidicolin for 48 h. The relative inhibition of increase in cell number by aphidicolin (%) was calculated as follows: [{(cell number at 48 h – cell number at 0 h) in the absence of aphidicolin – (cell number at 48 h – cell number at 0 h) in the presence of aphidicolin} ×100]/[(cell number at 48 h – cell number at 0 h) in the absence of aphidicolin]. If the cells did not grow at all in the presence of aphidicolin, then the (cell number at 48 h – cell number at 0 h) in the presence of aphidicolin becomes zero and the relative inhibition becomes 100%. However, sometimes in the presence of aphidicolin there were less number of cells at 48 h than at 0 h (instead of an increase in cell number), in which case the (cell number at 48 h – cell number at 0 h) becomes negative and the relative inhibition becomes more than 100%. (D) Comparison of γ-H2AX foci in wild-type, Arp8 OE, and Arp8 KO cells. The cells were treated with camptothecin (CPT) for 1 h, and after washing out the reagent, the cells were incubated without CPT for 2 h or 8 h. Immunostained γ-H2AX foci were observed under a fluorescence microsope, and the number of foci was counted using the ImageJ software. Plot below shows the number of γ-H2AX foci in the indicated cells relative to that in the camptothecin-untreated (-CPT) wild-type cells. (E) Wild-type, Arp8 OE, and Arp8 KO cells were treated with 1 µM camptothecin for 1 h. After washing out the reagent, the relative inhibition by camptothecin was shown as in C. Error bars indicate average mean ± SD (n =  at least 3 independent experiments).</p