A Specific Docking Site for DNA Polymerase α-Primase on the SV40 Helicase Is Required for Viral Primosome Activity, but Helicase Activity Is Dispensable

Abstract

Replication of simian virus 40 (SV40) DNA, a model for eukaryotic chromosomal replication, can be reconstituted in vitro using the viral helicase (large tumor antigen, or Tag) and purified human proteins. Tag interacts physically with two cellular proteins, replication protein A and DNA polymerase ␣-primase (pol-prim), constituting the viral primosome. Like the well characterized primosomes of phages T7 and T4, this trio of proteins coordinates parental DNA unwinding with primer synthesis to initiate the leading strand at the viral origin and each Okazaki fragment on the lagging strand template. We recently determined the structure of a previously unrecognized pol-prim domain (p68N) that docks on Tag, identified the p68N surface that contacts Tag, and demonstrated its vital role in primosome function. Here, we identify the p68N-docking site on Tag by using structure-guided mutagenesis of the Tag helicase surface. A charge reverse substitution in Tag disrupted both p68N-binding and primosome activity but did not affect docking with other pol-prim subunits. Unexpectedly, the substitution also disrupted Tag ATPase and helicase activity, suggesting a potential link between p68N docking and ATPase activity. To assess this possibility, we examined the primosome activity of Tag with a single residue substitution in the Walker B motif. Although this substitution abolished ATPase and helicase activity as expected, it did not reduce pol-prim docking on Tag or primosome activity on single-stranded DNA, indicating that Tag ATPase is dispensable for primosome activity in vitro.De novo DNA replication begins with RNA primer synthesis on single-stranded template DNA, followed by primer extension by a processive DNA polymerase. In prokaryotic replication, the activity of the primase is coordinated with unwinding of duplex DNA by a hexameric replicative helicase and a single-stranded DNA (ssDNA) 2 -binding protein, largely through dynamic physical interactions among the three proteins, which constitute a primosome (1-4). In eukaryotes, the DNA polymerase ␣-primase (pol-prim) complex catalyzes both RNA primer synthesis and extension, yielding RNA-DNA primers of 30 -35 nucleotides (5, 6). Unlike the single subunit prokaryotic primases, pol-prim is a stable heterotetramer composed of the primase heterodimer p48/p58, the catalytic DNA polymerase subunit p180, and a regulatory subunit (B or p68) (7). The eukaryotic replicative helicase complex, Cdc45/ Mcm2-7/GINS, and the ssDNA-binding protein, replication protein A (RPA), appear to coordinate primer synthesis by polprim with parental DNA unwinding, as in prokaryotes (8 -12). However, the nature of the eukaryotic primosome and its operation during chromosome replication, telomere maintenance, and checkpoint signaling at stalled replication forks remain elusive.Because pol-prim is essential for replication of simian virus 40 (SV40) DNA, we utilize this model system here to investigate the functional architecture of a eukaryotic primosome. SV40 DNA replication can be reconstituted in c...

Structural Basis for the Interaction of a Hexameric Replicative Helicase with the Regulatory Subunit of Human DNA Polymerase α-Primase

“…LTag unwinds the origin DNA, recruits RPA to the template, and then orchestrates RPA displacement by human Pol-prim for primer synthesis and extension. In a simpler reaction utilizing RPA-coated ssDNA as the template, LTag and Pol-prim are sufficient to reconstitute SV40 primosome activity even in the absence of LTag helicase activity (27)(28)(29)(30). These findings suggested that the key role of LTag in primosome activity may be to displace RPA and, in concert, position Pol-prim on the exposed ssDNA.…”

“…The interface on LTag consists of a hydrophobic patch centered on Tyr-552, Phe-617, and Met-621, flanked on one edge by a few positively charged residues. The LTag residue that had previously been implicated in p68N binding, Lys-425 (27), is not part of this interface (Fig. 3B and supplemental Fig.…”

“…Crystals were soaked stepwise in increasing concentrations of sodium malonate, pH 6.0 (1.25, 1.75, 2.25, and 2.75 M), for about 5 min at each step, and then flash-frozen in liquid nitrogen. (27,51). Each of the three structured domains is sufficient for its biochemical activity as follows: DnaJ chaperone domain (J), origin DNA binding domain (OBD), and helicase domain (HD), composed of the zinc (Zn) and ATPase subdomains, are boxed.…”

“…On the surface of p68N, a small hydrophobic patch flanked by acidic charges docks on LTag and is needed for SV40 primosome activity (23). An initial screen for a corresponding docking interface on LTag identified Lys-425 as important for docking and for primosome activity (27). However, the absence of a hydrophobic patch adjacent to Lys-425 on the LTag that could complement the docking surface on p68N was puzzling (27), raising questions about the role of Lys-425 in the p68-LTag binding interface and in primosome activity.…”

Initiation of DNA Replication from Closed Circular DNA

“…Its structure is not known, although the structure of the heterodimeric yeast pol alpha has been determined (Klinge, Nunez-Ramirez et al 2009). The human enzyme has been shown to bind T antigen (Dornreiter, Hoss et al 1990;Collins and Kelly 1991;Collins, Russo et al 1993;Dornreiter, Copeland et al 1993) and this interaction is needed for DNA replication (Taneja, Boche et al 2007); (Huang, Zhao et al 2010). Given its size, it might very well cover a substantial part of a hexamer.…”