FIG. 8.
Reactivation of dephosphorylated NS1O for helicase activity. Helicase assays were performed as described for Fig. 1B, using M13-VAR as a template. (A) Hydroxylapatite column fractions HA-0, HA-1, and HA-2 were tested for their intrinsic helicase activities (lanes 4 to 6) and for activation of the NS1O helicase function in the presence of the PKC cofactor Ca2++PS (lanes 8 to 10). Lane 7 shows the helicase activity of unstimulated NS1O. The NS1 mutant K405R served as a negative control (lane 3). The reaction products were analyzed by native 7% PAGE in the presence of 0.1% SDS. Lanes 1 and 2, native (NAT) and denatured (DEN) input DNA, respectively. (B and C) Characterization of PKC isoforms present within the HA-1 fraction, which is able to reactivate NS1O for helicase activity. (B) Dependence of HA-1-induced reactivation of NS1O helicase function on defined PKC cofactors. Atypical PKCs (ι/λ and ζ) are stimulated by acid lipids (PS) but are insensitive to phorbol esters such as TPA, whereas novel and classical PKCs are only slightly activated by PS alone but respond strongly to TPA. Dephosphorylated NS1O was assayed for helicase activity either alone (lane 5) or in the presence of HA-1 with or without the indicated PKC cofactors (lanes 7 to 9). The NS1 mutant K405R (lane 3) and native NS1P (lane 4) served as negative and positive controls, respectively. The effect of HA-1 in the absence of NS1 is shown in lane 6. Lanes 1 and 2, native and denatured input DNA, respectively. (C) Immunodetection of atypical PKC in hydroxylapatite fractions. Equal protein amounts of HA-1 and HA-2 were analyzed by Western blotting with αPKCι antibodies (Transduction Laboratories). A HeLa cell replication extract served as a positive control. Size markers and expected migrations of different PKC isoforms are indicated on the right (for abbreviations, see the legend to Fig. 7A). The estimated migrations of PKCλ/ι and PKCm, which are recognized by αPKCι, are indicated on the left.