Exploring the Sequence Sensitivity and Substrate Specificity of the Processive Viral Helicase Nph-II.

RNA and DNA helicases are ubiquitous enzymes involved in nearly every aspect of cellular nucleic acid metabolism across all organisms. Because nucleic acids play such a broad and fundamental role in all aspects of biology, understanding the diverse roles and mechanisms of the helicases that regulate their behavior is likewise of critical importance. This research focuses on the substrate specificity of the vaccinia viral helicase nucleoside triphosphate phosphohydrolase-II (NPH-II), a member of the helicase Superfamily 2 (SF2). NPH-II shows robust RNA helicase activity but typically displays little activity on DNA substrates; despite the fact that its nucleic acid-dependent ATPase activity is stimulated equally well by both RNA and DNA. Like other SF2 helicases, NPH-II is believed to make primary contacts with backbone residues of an RNA substrate, thus being insensitive to sequence variations of its substrate. Using a series of chemigenetic screens and modified substrates, we explore the sequence sensitivity of NPH-II and how it influences the substrate specificity and kinetic behavior of the enzyme. We report an unusual nucleobase bias, previously unreported in any SF1 or SF2 helicase, whereby purines are heavily preferred as components of both RNA and DNA tracking strands. The observed sequence bias confers to NPH-II robust DNA-RNA helicase activity. Using this sequence bias as a kinetic probe, we were also able to deduce several important features of the NPH-II unwinding mechanism. We show that contrary to previous assumptions, the rate of unwinding by NPH-II is not limited by initiation, but by a step that occurs late in the unwinding mechanism. These results indicate that in addition to ribose contacts, NPH-II utilizes the nucleobases to increase processivity on DNA tracks and to facilitate active unwinding of the substrate duplex. The sequence bias for NPH-II challenges the model of sequence neutrality invoked for processive helicases and the predominant backbone-tracking model of SF2 helicases. In addition to being generally applicable to related SF2 helicases, our findings have important implications for understanding the biological functions of NPH-II and how its activity may be targeted to specific cellular contexts. Informed by the requirements for efficient helicase activity in vitro and known biological functions, we suggest a biologically relevant role for sequence biased DNA-RNA helicase activity during viral transcriptional termination. Our results underscore the importance of accounting for in vivo substrate configurations, where possible, when designing biochemical experiments on helicase enzymes.