Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase

Adedeji, Adeyemi O.; Marchand, Bruno; te Velthuis, Aartjan J. W.; Snijder, Eric J.; Weiss, Susan; Eoff, Robert L.; Singh, Kamalendra; Sarafianos, Stefan G.

The coding region of nsp13 was amplified by PCR using 59BamHI-nsp13-ATGCTAGGATCCGCTGTAGGTGCTTGT-G-39 as the forward primer and 39XhoI-nsp13-GCTGACCTC-GAGTTATTGTAATGTAGCCACATTGC39 as the reverse primer. The PCR amplicons were digested with BamH I and XhoI followed by ligation into expression vector pGEX-4T-1 (Amersham Biosciences). After confirming the amplicon nucleotide sequences we used the flanking RsrII and XhoI restriction sites to subclone GST-nsp13 into the pFASTBAC1 vector (Invitrogen). In the final pFASTBAC1-GST-nsp13 construct, GST was fused at the N-terminus of nsp13. This plasmid was transformed into E. coli The kinetic parameters presented in this table are determined from the reaction mechanism presented in Figure 4A . Kinetic parameters are either a uncorrected or b corrected as described previously (48) by assuming that the last 10 base-pairs are unwound without direct helicase action. P is the processivity of DNA unwinding, L T is the total length of dsDNA and L 0 is the minimal length of dsDNA that is stable in the presence of active helicase and with last 10 bases unpaired. The kinetic step size m is defined as the (L T 2L 0 )/n, where n is the number of intermediates. doi:10.1371/journal.pone.0036521.t002 DH10Bac cells for transposition of GST-nsp13 from pFASTBAC1 into a bacmid, which is a baculovirus shuttle vector with a baculovirus-specific promoter (i.e, the polyhedrin or p10 promoter). The bacmid was propagated in E. coli DH10Bac as a large plasmid that confers resistance to kanamycin and can complement a lacZ deletion present on the chromosome to form blue (Lac+) colonies in the presence of a chromogenic substrate such as Bluegal or X-gal and the inducer IPTG. Insertion of GST-nsp13 into the bacmid disrupted expression of lacZa, allowing the selection of GST-nsp13-containing bacmids (white colonies) and isolation of DNA using the High Purelink DNA isolation kit (Invitrogen). Following confirmation of GST-nsp13 gene insertion in the bacmid, the recombinant bacmid was then transfected into Table 3 . Kinetic parameters for the effect of nsp12 on the unwinding activity of GST-nsp13. MBP-nsp13 Cloning, Expression, and Purification nsp13 was cloned into the pMAL-p4x using BamHI and XbaI at the 59 and 39 ends. The resulting pMal-nsp13 was used to transform E. coli TB1 cells (New England Biolabs). MBP-nsp13 was expressed and purified by amylose affinity chromatography (New England Biolabs). MBP-nsp13 was further purified by sizeexclusion chromatography on a Superdex-200 10/300GL column (GE Healthcare) run under isocratic conditions with 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 5% glycerol. Fractions containing pure nsp13 were concentrated and stored at 280uC.