The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases - PubMed (original) (raw)

Purification and DNA-unwinding activity of the SARS coronavirus NTPase/helicase.Panel A, purification of the SARS-CoV Hel as observed by 4–12% SDS-PAGE. Lane 1, marker, from bottom (in kilodaltons) 10, 15, 20, 25, 30, 40, 50 (strong), 60, 70, 80, 90, 100, 120, 160, 220. Lane 2, Whole cell extract from E. coli BL21 (DE3) expressing SARS-CoV Hel (5 μg). Lane 3, soluble extract from E. coli BL21 (DE3) expressing SARS-CoV Hel (10 μg). Lane 4, SARS-CoV Hel after purification by nickel affinity chromatography (1.5 μg). Lane 5, SARS-CoV Hel after purification on Red Sepharose CL-6B (0.4 μg). Panel B, schematic showing the fully double-stranded (dB) and partially double-stranded DNA substrates (d5T = 5′-oligo(dT)20 tail, d3T = 3′-oligo(dT)20 tail) used in the unwinding assay. An asterisk indicates the position of the 32P radiolabel. Panel C, the SARS-CoV Hel unwinds duplex DNA with a 5′ to 3′ polarity. The positions of the intact dsDNA duplexes (dB, d5T, and d3T), as well as the released (labeled) ssDNA strand on the polyacrylamide gel, are indicated with arrows. Lanes 1–5, reactions containing 30 fmol of DNA duplex; lanes 2–5, reactions containing 6 pmol of helicase; lanes 1–4, reactions containing 2.5 m

m

ATP; lane 1, the d5T DNA duplex was heat-denatured. Panel D, titration of helicase concentration. Lanes 1–4, reactions containing 150 fmol, 600 fmol, 1.5 pmol, and 3 pmol of helicase, respectively, in addition to 30 fmol of d5T DNA duplex and 2.5 m

m

ATP.