Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding

Abstract

Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N-terminal portion, but the helicase domain is solely responsible for the enzyme's unwinding activity. In addition, we demonstrate a novel stimulation of RecQ4's helicase activity by replication protein A, similar to that of RecQ1, BLM, WRN, and RecQ5. Together, these data indicate that specific biochemical activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases.

Figure 1. Purified RecQ4 possesses helicase activity

A) Coomassie blue stained SDS-PAGE gel of purified wild-type (WT) RecQ4 (250 ng ; lane 2). B) The helicase activity of purified wild-type RecQ4 (10, 20, 50, 100, and 200 nM) was assayed on 0.5 nM fork-1 (T₁:B₁; upper panel) and fork-2 (T₂:B₂; lower panel) in the absence (lanes 2-6) and presence (lanes 8-12) of unlabeled single-stranded primers (12.5 nM) T₁ or T₂, respectively. The Δ (lane 13) indicates heat denatured substrate. Asterisk indicates the position of the radiolabel. C) The strand annealing activity of RecQ4 (5, 10, 20, and 50 nM) measured with the radiolabeled ssDNA T₁ and T₂ and their complimentary primers B₁ (lanes 2-5) and B₂ (lanes 7-10), respectively. D) Quantitation of RecQ4's strand annealing. The values represent an average of four independent experiments corrected for substrate annealing in the absence of RecQ4. Error bars represent standard deviation.

Figure 2. Helicase activity is intrinsic to RecQ4

A) Diagram of the SFII helicase domain of RecQ4. Amino acid residues surrounding the conserved lysine 508 (bold) are noted for the wild-type protein. The point mutation K508M (KM) is indicated in bold italics. B) Coomassie blue stained SDS-PAGE gel of purified K508M mutant (KM; lane 2) and wild-type (WT; lane 3) RecQ4 (250 ng/lane). C) The strand annealing activity of KM RecQ4 (5, 10, 20, and 50 nM; lanes 3-6) versus WT (50 nM; lane 2) measured with the radiolabeled ssDNA T₁ and its complimentary primer B₁. D) Quantitation of RecQ4 strand annealing. The values represent an average of four independent experiments corrected for substrate annealing in the absence of RecQ4. The WT data is taken from Figure 1D. Error bars represent standard deviation. E) The helicase activity of KM (50, 100, and 200 nM; lanes 2-4) versus WT (200 nM ; lane 5) RecQ4 assayed on fork-2 (T₂:B₂). The Δ (lane 6) indicates heat denatured substrate. Asterisk indicates the position of the radiolabel. F) ATP hydrolysis by KM (2, 20, and 200nM; lanes 2-4) versus WT RecQ4 (2, 20, and 200nM; lanes 5-7) in the presence of ssDNA co-factor evaluated by thin-layer chromatography.

Figure 3. RecQ4 helicase activity is ATPase-dependent and sequence-independent

The helicase activity of RecQ4 (100 and 200 nM) was measured on fork-1 (T₂:B₂; upper panel) and fork-3 (T₃:B₃; lower panel) in the presence of 5 mM ATP (lanes 2-3), 5 mM non-hydrolyzable ATPγS (lanes 4-5), or absence of ATP (lanes 6-7). The Δ (lane 8) indicates heat denatured substrate. Asterisk indicates the position of the radiolabel.

Figure 4. RPA stimulates RecQ4 helicase activity

A) The helicase activity of RecQ4 (34 nM ; lane 2) was measured on fork-2 (T₂:B₂; upper panel) and fork-3 (T₃:B₃; lower panel) in the presence of increasing concentrations of RPA (0.5, 1, 2, and 5 nM; lanes 3-6). The Δ (lane 11) indicates heat denatured substrate. Asterisk indicates the position of the radiolabel. B) Quantitation of RecQ4 unwinding in the presence of RPA. The values represent an average of four independent experiments corrected for destabilization of the substrate by RPA in the absence of RecQ4. Error bars represent standard deviation. C) The helicase activity of WRN (0.125 nM and 0.5 nM; lane 2 upper and lower panels, respectively) was measured on fork-2 (T₂:B₂; upper panel) and fork-4 (T₄:B₄; lower panel) in the presence of increasing concentrations of RPA (1, 2, and 5 nM, lanes 3-6 upper panel; 5, 10, and 20 nM lanes 3-6 lower panel). The Δ (lane 11) indicates heat denatured substrate. Asterisk indicates the position of the radiolabel. D and E) Quantitation of WRN unwinding of fork-2 and fork-4, respectively, in the presence of RPA. The values represent an average of three independent experiments corrected for destabilization of the substrate by RPA in the absence of WRN. Error bars represent standard deviation.