Nucleic Acid Unwinding by Hepatitis C Virus and Bacteriophage T7 Helicases Is Sensitive to Base Pair Stability

Yong-Joo Jeong,Ilker Donmez,Smita S. Patel,Vaishnavi Rajagopal

doi:10.1074/jbc.m702136200

Abstract

Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.

Highlights

Helicases are proteins that translocate in one specific direction along the nucleic acid backbone to catalyze processes such as the separation of the complementary strands of a double helical nucleic acid, recombination of DNA molecules, or remodeling of proteins bound to nucleic acids [1,2,3,4,5]
A member of the non-ring-shaped helicases that we have studied here is encoded by the hepatitis C virus (HCV)
The results clearly show that the unwinding rate depends on the sequence and decreases as the average base pair stability of the unwinding substrates increases

Summary

EXPERIMENTAL PROCEDURES

Assays with T7 helicase were in Buffer T (50 mM Tris-Cl, pH 7.6, 40 mM NaCl, 10% glycerol) at 18 °C. Assays with HCV helicases were in Buffer H (50 mM MOPS-Na, pH 7.0, 5 mM MgCl2, 5 mM dithiothreitol, 0.1% Tween 20) at 22 °C. The length of the dsNA region of the substrates was chosen carefully; it cannot be too short, because at room temperature 8 –16 bp of dsNA of a particular sequence can have ⌬G close to zero and can get spontaneously unwound [29, 42]. The length of the dsNA region cannot be too long, otherwise the unwound strands may reanneal behind the helicase during the reaction [29]. The range of GC content in the dsNA provided us with unwinding substrates of increasing overall stability.

Unwinding substrates and their properties

RESULTS

DISCUSSION