Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A

Abstract

RecQ helicases are required for the maintenance of genome stability. Characterization of the substrate specificity and identification of the binding partners of the five human RecQ helicases are essential for understanding their function. In the present study, we have developed an efficient baculovirus expression system that allows us to obtain milligram quantities of recombinant RECQ1. Our gel filtration and dynamic light scattering experiments show that RECQ1 has an apparent molecular mass of 158 kDa and a hydrodynamic radius of 5.4 +/- 0.6 nm, suggesting that RECQ1 forms dimers in solution. The oligomeric state of RECQ1 remains unchanged upon binding to a single-stranded (ss)DNA fragment of 50 nt. We show that RECQ1 alone is able to unwind short DNA duplexes (<110 bp), whereas considerably longer substrates (501 bp) can be unwound only in the presence of human replication protein A (hRPA). The same experiments with Escherichia coli SSB show that RECQ1 is specifically stimulated by hRPA. However, hRPA does not affect the ssDNA-dependent ATPase activity of RECQ1. In addition, our far western, ELISA and co-immunoprecipitation experiments demonstrate that RECQ1 physically interacts with the 70 kDa subunit of hRPA and that this interaction is not mediated by DNA.

Figure 1

SDS–PAGE analysis of RECQ1 gene expression in insect sf9 cells. Lane 1, low range molecular mass markers (in kDa); lane 2, lysate of sf9 cells infected with recombinant RECQ1 baculovirus; lane 3, washes of TALON metal affinity resin with 500 mM NaCl and 12. 5 mM imidazole; lane 4, elution from TALON metal affinity resin with 100 mM NaCl and 120 mM imidazole. The gel was 10% SDS–polyacrylamide stained with Coomassie blue R250. Lane 4 contained 3.75 µg of purified recombinant RECQ1.

Figure 2

Gel filtration and dynamic light scattering analysis. (A) Gel filtration experiments were performed as described in Materials and Methods. The TSK-GEL G3000SW_XL column was calibrated with thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (45 kDa), myoglobin (17 kDa) and vitamin B₁₂ (1.35 kDa). The amount of RECQ1 loaded was 37.5 µg. The solid curve shows the elution profile of RECQ1 and the dashed curve the elution profile of BSA (66 kDa). (B) Normalized auto-correlation function (ACF) for control BSA (solid circles), RECQ1 alone (open circles) and RECQ1 + 50mer ssDNA + ATP + Mg²⁺ (open squares). Measurements were carried out at room temperature (23 ± 0.1°C) with a protein concentration of ∼2.5 µM in buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM DTT). Solid lines are the fit with the built-in regularization method (Dynamics v6.0 from Protein Solutions). Radii were 3.5 ± 0.4 nm for BSA and 5.4 ± 0.6 nm for all the RECQ1 samples. (Insert) Band shift assay with the 50 nt ssDNA probe in the absence (1) and presence (2) of RECQ1. The experiments were carried out with 1 µM RECQ1 and 0.4 nM DNA as described in Materials and Methods.

Figure 3

Unwinding studies with DNA substrates of increasing duplex length. Helicase assays were performed as described in Materials and Methods. The indicated amount of RECQ1 was used to unwind the M13mp18 partial duplex substrates of 17 (black diamond), 25 (open square), 50 (black circle), 110 (black triangle) and 216 bp (open triangle). The concentration of the substrate was always 0.4 nM. After incubation at 37°C for 30 min, the reaction mixtures were resolved on a 12% non- denaturing polyacrylamide gel.

Figure 4

Stimulation of RECQ1 helicase activity on a 216 bp duplex substrate by hRPA and ESSB. (A) The indicated amount of hRPA (filled circle) or ESSB (filled square) was preincubated with 216 bp duplex DNA substrate (0.4 nM). The reactions were initiated by adding 300 nM RECQ1 and carried on at 37°C for 45 min. The reaction mixtures were resolved on a 6% non-denaturing polyacrylamide gel. The concentrations (nM) of hRPA and ESSB are indicated above each lane in the autoradiogram. Lanes C and D are control assays without enzyme and with heat-denatured substrate. The graph shows the percentage of unwinding versus the concentration of single-stranded binding protein. (B) Kinetics of unwinding of the 216 bp duplex DNA substrate by RECQ1 (300 nM) in the presence of hRPA (300 nM). The experiment was performed using the same reaction conditions as described above. At the indicated times (min), 20 µl of the reaction mixture were removed and quenched with 50 mM EDTA. The mixtures were resolved on a 6% non-denaturing polyacrylamide gel.

Figure 5

Unwinding studies with DNA substrates of increasing duplex length in the presence of hRPA. The indicated amount of hRPA was preincubated with different DNA substrates containing partial duplexes of 301 (filled diamond), 416 (open square), 501 (filled circle), 603 (open triangle) and 807 bp (filled square). The concentration of the substrate was always 0.4 nM. The reactions were initiated by adding 300 nM RECQ1. The reaction was incubated at 37°C for 120 min. The reaction mixtures were resolved on a 6% non-denaturing polyacrylamide gel. The percentage of unwinding is expressed as a function of hRPA concentration. The hRPA concentrations (nM) are indicated above each lane in the autoradiogram. Lanes C and D are control assays without enzyme and with heat-denatured substrate.

Figure 6

Analysis of the ATPase activity of the recombinant RECQ1 and effect of hRPA on rate of ATP hydrolysis. Filled circles, reactions with M13mp18; open circles, reactions with M13mp18 and hRPA (50 nM); filled squares, reactions with 25 nt ssDNA; open squares, reactions with 25 bp ssDNA and hRPA (50 nM); filled triangles, reactions with 17 bp ssDNA. The initial velocities for ATP hydrolysis were expressed as a function of the ATP concentration. The experimental points were fitted to the Michaelis–Menten equation: V₀ = V_maxX/(K_m +X), where V₀ is the initial velocity and X is the substrate concentration (ATP). Each value represents the mean of at least five independent measurements.

Figure 7

RECQ1 physically interacts with the 70 kDa subunit of hRPA. (A) Detection of the RECQ1–hRPA complex by far western analysis. Purified recombinant RECQ1, BSA, hRPA, and Ku were subjected to SDS–PAGE. I, Coomassie blue staining of the gel; II, proteins were transferred to a Hybond-P membrane and then incubated with recombinant RECQ1, western blotting using anti-6His monoclonal antibody then being used to detect the presence of RECQ1 on the membrane; III, in control experiments Hybond-P membrane containing hRPA was incubated with buffer alone to verify that there was no cross-reactivity between the anti-6His monoclonal antibody and hRPA. (B) Detection of RECQ1–hRPA complex by ELISA. RECQ1-coated wells (18 nM application) were incubated with increasing amounts of hRPA protein for 1 h at 30°C. Wells were aspirated and washed three times and bound hRPA protein was detected by ELISA using a mouse monoclonal antibody against hRPA (70 kDa subunit) protein. Absorbance readings at each point were corrected by subtracting a background A₄₉₀ reading generated with BSA-coated wells.

Figure 8

Co-immunoprecipitation of RECQ1 and hRPA from human nuclear extracts with and without ethidium bromide. RECQ1 antibody co-precipitates RECQ1 and hRPA from HeLa nuclear extracts both in the presence and absence of ethidium bromide. The blot was probed with mouse anti-RPA antibody. Lane 1, purified hRPA (250 ng); lane 2, HeLa nuclear extract (54 µg); lane 3, immunoprecipitate from HeLa nuclear extract (1.36 mg) with ethidium bromide (50 µg/ml) using rabbit anti-RECQ1 antibody; lane 4, immunoprecipitate from HeLa nuclear extract (1.36 mg) using rabbit anti-RECQ1 antibody; lane 5, control precipitate from HeLa nuclear extract (1.36 mg) using normal rabbit IgG.