Expression and purification of the recombinant MBP-tagged host proteins and the MBP-tagged TBSV p33 and p92 replication proteins and Ded1p helicase from E. coli were carried out as described earlier with modifications [71] . Briefly, the expression plasmids were transformed separately into E. coli strain BL21(DE3) CodonPlus. Protein expression was induced using isopropyl b-Dthiogalactopyranoside (IPTG) for 8 h at 23uC in the case of host proteins and at 16uC in the case of p33 and p92, then the cells were collected by centrifugation (5,000 rpm for 5 min). The cells were suspended and sonicated in MBP column buffer containing 30 mM HEPES-KOH pH 7.4, 25 mM NaCl, 1 mM EDTA, 10 mM b-mercaptoethanol. The extract was then centrifuged at 16,000 g for 10 min, followed by incubation with amylose resin (NEB) for 15 min at 4uC. After washing the resin 2 times with the column buffer, the proteins were eluted with column buffer containing 0.18% (V/W) maltose. Purification of GST-tagged TDH2 (pGEX-TDH2) [50] was carried out using glutathione resin and eluted with 10 mM glutathione, 10 mM ß-mercaptoethanol in the column buffer following the same protocol as MBPproteins. Eluted proteins were aliquoted for storage at 280uC. Protein fractions used for the replication assays were at least 95% pure, as determined by SDS-PAGE (not shown). We have previously shown that our Ded1p preparation has helicase activity on short RNA/DNA duplexes in vitro [48] .