VACCINIA VIRUS-RNA HELICASE - NUCLEIC-ACID SPECIFICITY IN DUPLEX UNWINDING

Vaccinia virus RNA helicase (NPH-II) catalyzes nucleoside triphosphate -dependent unwinding of duplex RNAs containing a single-stranded 3' RN A tail. In this study, we examine the structural features of the nucle ic acid substrate that are important for helicase activity. Strand dis placement was affected by the length of the 3' tail. Whereas NPH-II ef ficiently unwound double-stranded RNA substrates with 19- or 11-nucleo tide (nt) 3' tails, shortening the 3' tail to 4 nt reduced unwinding b y an order of magnitude. Processivity of the helicase was inferred fro m its ability to unwind a tailed RNA substrate containing a 96-bp dupl ex region. NPH-II exhibited profound asymmetry in displacing hybrid du plexes composed of DNA and RNA strands. A 34-bp RNA-DNA hybrid with a 19-nt 3' RNA tail was unw;ound catalytically, whereas a 34-bp DNA-RNA hybrid containing a 19-nt 3' DNA tail was 2 orders of magnitude less e ffective as a helicase substrate. NPH-II was incapable of displacing a 34-bp double-stranded DNA substrate of identical sequence. 3'-Tailed DNA molecules with 24- or 19-bp duplex regions were also inert as heli case substrates. On the basis of current models for RNA-DNA hybrid str uctures, we suggest the following explanation for these findings. (i) Unwinding of duplex nucleic acids by NPH-II is optimal when the polynu cleotide strand of the duplex along which the enzyme translocates has adopted an A-form secondary structure, and (ii) a B-form secondary str ucture impedes protein translocation through DNA duplexes.