FIG. 4.

Characterization of recombinant PIF subunits expressed from separate baculovirus vectors. (A) SDS-PAGE analysis of purified recombinant PIF. DNA-affinity purified PIF from HeLaS3 cells was compared to purified recombinant PIF p96-p79, expressed in insect cells by using baculovirus vectors. The positions of the p96 and p79 subunits are indicated by arrows. (B) DNase I protection and methylation interference patterns of rPIF binding to a duplex DNA fragment containing the minimal replication origin, in which the top strand, as represented in Fig. 1, was ³²P labeled at its 3′ end. The sequence through this part of the origin is indicated on the right. Lowercase letters indicate flanking vector DNA sequence. PIF half-sites are shaded, the consensus ATF site is boxed, bubble nucleotides are shown in outline, and the open-ended box indicates the start of the NS1 footprint, in which the first TGGT motif is stippled. Sequences protected by rPIF from DNase I digestion are shown in the left lanes. The right three lanes, labeled MI, show methylation interference profiles: input indicates piperidine cleavage products of the methylated DNA sample used in the binding assay; bound indicates cleaved DNA from the retarded rPIF-DNA complex; and free indicates free probe which was not shifted by the binding reaction. Methylated residues which impair rPIF binding are indicated by arrows.