FIG 6.

Conserved regions in PUBS mediate its interaction with UAF1 and its DNA replication stimulatory activity. (A) Schematic representation of the PUBS-E1Δ chimeric helicase with the PUBS region enlarged underneath. The highly conserved N- and C-terminal regions of PUBS are diagrammed as black boxes labeled N and C, respectively. Amino acid boundaries are indicated. Also shown is the amino acid sequence of the N and C regions in which the motifs that were deleted to create the MutN and MutC mutations are underlined. (B) Coimmunoprecipitation of endogenous UAF1 with the WT and mutant PUBS-E1Δ proteins. C33A cells were transfected with the indicated 3F-E1 expression plasmids, and the encoded protein was immunoprecipitated with an anti-Flag antibody. The presence of UAF1 in the immunoprecipitates (IP) and input cell extracts (IN) was determined by Western blotting with an anti-UAF1 antibody. Wild-type E1 and the E1Δ protein were used as a positive and negative control, respectively. (C) DNA replication activities of wild-type and mutant PUBS-E1Δ proteins. DNA replication activities, presented as RLuc/FLuc ratios, were measured in cells transfected with increasing amounts of the indicated E1 expression vector (2.5, 5, 10, and 25 ng). The E1Δ protein was used as a negative control. Each value represents the average from two independent experiments, each performed in triplicates, with the standard deviation indicated by an error bar.