Single strand binding proteins increase the processivity of DNA unwinding by the hepatitis C virus helicase

Abstract

The nonstructural NS3 protein of the hepatitis C virus is a multifunctional enzyme with an N-terminal serine protease activity and a C-terminal helicase activity. The helicase is capable of unwinding both DNA and RNA duplexes; however, the overall processivity of the helicase is fairly low. We show here that single-strand binding (SSB) proteins enhance the unwinding processivity of both the NS3 helicase domain (NS3h) and the full-length protease-helicase NS3-4A. The detailed study of the effect of SSB on the DNA unwinding activity of NS3h indicates that the SSB stabilizes the helicase at the unwinding junction and prevents its dissociation. These results suggest a potential role for either cellular or virus-encoded SSB protein in improving the processivity of the NS3 in vivo.

Fig. 1

HCV NS3h has a low intrinsic processivity of DNA unwinding. (A) Unwinding kinetics of a 40bp forked DNA substrate (Table I, 3) under single turnover conditions. NS3h (200nM) was incubated with 5 nM ss/dsNA substrate in buffer in one syringe, and mixed with equal volume of 10 mM ATP, 1μM PolyU and 400nM DNA trap in the same buffer from the other syringe, for the times indicated. The unwinding reaction was stopped by the addition of a 1.5 fold volume of quenching solution. The reactions were carried out in a rapid quenched-flow instrument. NS3h unwinds less than 1% of the total DNA. (B) Unwinding kinetics of the 18bp forked DNA substrate (Table I, 6) under of single turnover conditions. Reactions were performed as explained above, but a 18bp forked DNA substrate was used instead of the 40bp forked DNA. NS3h unwinds about 80% of the 18bp substrate with a rate of 3.7 bp/s. (C) Unwinding kinetics of the 40bp forked DNA substrate under multiple turnover conditions. Unwinding reactions were carried out by incubating 200nM of NS3h with 5 nM ss/dsNA substrate in buffer in one syringe, and mixing with equal volume 10 mM ATP but no trap, in the same buffer from the other syringe for the times indicated. NS3h unwinds about 60% of the substrate at a rate of 0.4 bp/s.

Fig. 2

DNA unwinding processivity of NS3h unwinding is enhanced by single strand binding proteins. (A) The 40bp fork substrate and the unwound single stranded DNA (resolved by native PAGE) after various times of reaction with NS3h in the presence of E.coli SSB. NS3h (200nM) was incubated with 5 nM ss/dsNA substrate in buffer in one syringe, and mixed with equal volume of 10 mM ATP, 2μM E.coli SSB in the same buffer from the other syringe, for the times indicated (B) Fraction of the 40bp DNA unwound in the presence of E. coli SSB ( ) or T7 SSB, gp2.5 ( ) as a function of reaction time. NS3h (200nM) was incubated with 5 nM ss/dsNA substrate in buffer in one syringe, and mixed with equal volume of 10 mM ATP, 2μM E.coli SSB or 10μM T7 SSB (gp2.5) in the same buffer from the other syringe, for the times indicated. The unwinding rates range between 3.4 bp/s and 3.7 bp/sec and the amount of products formed range between 83–87% in the presence of E. coli SSB. The unwinding rate in the presence of gp2.5 ranges between 5.7bp/sec and 5.9 bp/sec and the amount of products formed range between 36%–42%.

Fig. 3

Effect of the 5′-ssDNA tail on the unwinding efficiency of NS3h in the presence of SSB. The unwinding reaction by NS3h was carried out in the presence of SSB using substrates that either possessed the 5′-tail (Table I, 3) or lacked it (Table I, 4). NS3h (200nM) was incubated with 5 nM ss/dsNA substrate in buffer in one syringe, and mixed with equal volume of 10 mM ATP, 2μM E.coli SSB in the same buffer from the other syringe, for the times indicated The fraction of DNA unwound in each case was plotted as a function of reaction time. The 40bp substrate with the 5′-tail was unwound to a greater extent ( ) than the DNA with out the 5′-tail ( ), but with similar DNA unwinding rates in the range of 3.4 – 3.7 bp/s and 2.9 – 3.1 bp/s, respectively.

Fig. 4

Mechanism of SSB action –DNA re-annealing trap. Experiments designed to investigate if SSB was preventing DNA re-annealing behind the helicase. (A) Schematic representation of the unwinding reaction in A-1 shows that the SSB tetramers (in green) bind to the displaced strand and prevent DNA re-annealing as NS3h molecules (in red) unwind the DNA. The unwinding reaction in A-2 shows that an excess of the DNA reannealing trap (strand complementary to the displaced strand) prevent DNA re-annealing. (B) The kinetics of 40bp substrate unwinding in the presence of SSB ( ) and in the presence of the DNA re-annealing trap ( ). (C) Fraction of DNA unwound in the presence of an increasing amount of the DNA re-annealing trap, from 1 – 500 μM (black bar) compared to the reaction with SSB (grey bar).

Fig. 5

Mechanism of SSB action - mutant poisoning experiments. (A) Wild-type NS3h ( ) and mutant D261A NS3h ( ) bind to single-stranded DNA (Table I, 6) with equal affinity as assessed by the nitrocellulose filter binding assays. 20nM of 25mer DNA was incubated with increasing concentrations of either WT-NS3h or mutant D261A NS3h for 30 min at room temperature. The fraction of protein bound was plotted as a function of protein concentration. (B) Schematic representation of the unwinding experiments done in the presence of the mutant D261A. The red circles represent the WT NS3h, while the blue circles (on the right) represent the mutant D261A. The green tetramer represents the SSB protein. In the mutant poisoning experiments, The wild-type enzyme is pre-incubated with the DNA substrate and the reaction is initiated with the SSB-mutant NS3h-ATP mixture. The reaction is then quenched with SDS-EDTA quenching solution after appropriate times. (C) The unwinding of the 40bp substrate was measured using a constant amount of WT NS3h (100 nM) and increasing amounts of the mutant NS3h in the absence ( ) or in the presence of SSB ( ). 5nM forked substrate was pre-incubated with 200nM WT-NS3h. The unwinding reaction was initiated by mixing with equal volume of 10mM ATP and increasing concentrations of mutant D261A, either in the presence or absence of 1μM E.coli SSB. The amount of products formed in each case was plotted as a function of increasing mutant concentration. (D) The kinetics of 40bp substrate unwinding by NS3h in the presence of SSB and excess of the mutant D261A. WT-NS3h (200nM) was incubated with 5 nM ss/dsNA substrate in buffer in one syringe, and mixed with equal volume of 10 mM ATP, 2μM E.coli SSB and 1μM mutant NS3h D261A, in the same buffer from the other syringe for the times indicated. About 40% of the substrate is unwound at a rate of 3.9 bp/s.

Fig. 6

Unwinding activity of the NS3h monomer. (A) Unwinding of the 30bp substrate with a dT₅ 3′-loading tail and dT₃₅ 5′-tail (Table I, 5) by NS3h was carried out with in the presence of SSB and excess of the mutant NS3h. The fraction of substrate unwound is plotted as a function of time. About 45% of the substrate is unwound by NS3h at an average rate 4 bp/s. (B) Unwinding reaction was carried out using 10 nM NS3h and 10 nM 30bp forked DNA substrate. About 12% of the DNA is unwound (1.8 bp/s) in the burst phase, which is very close to the expected 16% (1/6^th of the total enzyme, since the average enzyme binding site on the DNA is 7 bases).

Fig. 7

SSB enhances the unwinding processivity of the full length helicase-protease NS3-4A. The 40bp forked substrate (Table I, 3) is unwound by the full length helicase-protease NS3-4A in the presence ( ) and absence ( ) of SSB at average rates of 0.22 bp/s and 0.4 bp/s, respectively.