Lane 1 shows 7.5% of the LTag input used for pulldowns in lanes 2–7. C and D, FLAG beads without ( lane 2) or with bound p48/His 6-FLAG×2-p58 heterodimer ( C) or SJK237-31-Sepharose beads without ( lane 2) or with bound p180 ( D) were incubated with soluble, purified full-length WT or the point mutant LTags as indicated. Lane 1, 15% of the LTag input used for pulldowns in lanes 2–7. Retained proteins were analyzed by Western blot with the indicated antibodies. B, glutathione-agarose beads bound to either GST ( lane 2) or GST-p68N ( lanes 3–7) were incubated with full-length WT or the indicated point mutant LTags. A, purified full-length WT and the indicated mutant LTags were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Specific role of LTag-p68 interaction in primosome activity. Lanes 1 and 2 contain unboiled ( UB) and boiled ( B) DNA substrate. E, helicase activities of LT108 mutants that have disrupted p68N binding were assayed over a time course of 30 s to 40 min. Lane 1, 15% of the LT108 input amounts used in lanes 2–7. Retained proteins were visualized by Western blot with the indicated antibodies. D, glutathione-agarose beads bound to either GST ( lane 2) or GST-p53 DBD ( lanes 3–7) were incubated with LT108 WT or substitution proteins as indicated. C, binding interfaces on LTag for p53 ( red) (39) and for p68 ( blue) (Fig. For the pulldown assays in A and B, the input LTag for initial incubation for each lane was 100 μg (see “Experimental Procedures”). Lane 1, LTag retained on Ni resin in the absence of His 6-p68N lane 2, LTag retained on His 6-p68N-bound Ni resin lanes 3–8, LTags retained on Ni resin bound to mutant His 6-p68Ns (as marked). B, pulldown assays were performed to evaluate the effect of p68N mutations of the residues within the interface on binding of LTag. Lane 1, LTag helicase domain ( LT-HD) retained on Ni resin in the absence of His 6-p68N lane 2, the LTag retained on His 6-p68N-bound Ni resin lanes 3–11, mutant LTags (as marked) retained on His 6-p68N-bound Ni resin. A, pulldown assays were performed to evaluate the effect of LTag mutations of the residues within the interface on binding to p68N. Mutational analysis of the LTag-p68N interface and functional validation. C and D, close-up views of the detailed LTag-p68N interactions within region 1 ( C) and region 2 ( D), showing the charge-charge interactions in region 1 and the hydrophobic interactions in region 2, respectively. The LTag Lys-425 is not part of the p68N-binding residues, but it is located immediately next to the interface. The residues involved in the interface contacts are colored as follows: hydrophobic residues in yellow, positively charged residues in blue, and negatively charged residues in red. B, surface representation of LTag ( bottom) and p68N ( top), showing the interface areas on both proteins. Two regions featuring hydrophobic and electrostatic interactions are indicated. Secondary structures involved in the interaction of both proteins are labeled for LTag and p68N, respectively. LTag domains D1, D2, and D3 are indicated. A, ribbon illustration of the complex structure of one LTag molecule (in cyan) binding to one p68N (in green). Here, we report the detailed large T antigen-p68 interface, as revealed in a co-crystal structure and validated by site-directed mutagenesis, and we demonstrate its functional importance in activating the SV40 primosome in cell-free reactions with purified Pol-prim, as well as in monkey cells in vivo.ĭetailed LTag-p68N interface interactions. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. At least three subunits of Pol-prim interact physically with the hexameric replicative helicase SV40 large T antigen, constituting a simple primosome that is active in vitro. Pol-prim is thought to interact with eukaryotic replicative helicases, forming a dynamic multiprotein assembly that displays primosome activity.
Pol-prim is composed of a primase heterodimer that synthesizes an RNA primer, a DNA polymerase subunit that extends the primer, and a regulatory B-subunit (p68) without apparent enzymatic activity. DNA polymerase α-primase (Pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand.
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