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

Open AccessPublished:August 03, 2010DOI:https://doi.org/10.1074/jbc.M110.156240
      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 α-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.

      Introduction

      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)
      The abbreviations used are: ssDNA
      single-stranded DNA
      AAA+
      ATPases associated with various cellular activities
      OBD
      origin DNA-binding domain
      Pab101
      polyomavirus monoclonal antibody 101
      pol-prim
      DNA polymerase α-primase
      RPA
      replication protein A
      Tag
      large tumor antigen
      P4
      patch 4 mutant.
      -binding protein, largely through dynamic physical interactions among the three proteins, which constitute a primosome (
      • Corn J.E.
      • Berger J.M.
      ,
      • Hamdan S.M.
      • Richardson C.C.
      ,
      • Langston L.D.
      • Indiani C.
      • O'Donnell M.
      ,
      • Marians K.J.
      ). In eukaryotes, the DNA polymerase α-primase (pol-prim) complex catalyzes both RNA primer synthesis and extension, yielding RNA-DNA primers of 30–35 nucleotides (
      • Copeland W.C.
      • Wang T.S.
      ,
      • Kuchta R.D.
      • Stengel G.
      ). 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) (
      • Kunkel T.A.
      • Burgers P.M.
      ). The eukaryotic replicative helicase complex, Cdc45/Mcm2–7/GINS, and the ssDNA-binding protein, replication protein A (RPA), appear to coordinate primer synthesis by pol-prim with parental DNA unwinding, as in prokaryotes (
      • Ilves I.
      • Petojevic T.
      • Pesavento J.J.
      • Botchan M.R.
      ,
      • Gambus A.
      • van Deursen F.
      • Polychronopoulos D.
      • Foltman M.
      • Jones R.C.
      • Edmondson R.D.
      • Calzada A.
      • Labib K.
      ,
      • Aparicio T.
      • Guillou E.
      • Coloma J.
      • Montoya G.
      • Méndez J.
      ,
      • Tanaka T.
      • Nasmyth K.
      ,
      • Walter J.
      • Newport J.
      ). 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 cell-free reactions with purified recombinant human proteins and the viral large T antigen (Tag) (
      • Waga S.
      • Stillman B.
      ). Tag serves as the replicative helicase and orchestrates the assembly of the viral replisome. Tag monomers first assemble cooperatively into a preinitiation complex on the viral origin of DNA replication, forming two hexamers oriented head-to-head, akin to the Mcm2–7 (minichromosome maintenance 2–7) hexamer assemblies recently visualized on yeast origins (
      • Cuesta I.
      • Núñez-Ramírez R.
      • Scheres S.H.
      • Gai D.
      • Chen X.S.
      • Fanning E.
      • Carazo J.M.
      ,
      • Evrin C.
      • Clarke P.
      • Zech J.
      • Lurz R.
      • Sun J.
      • Uhle S.
      • Li H.
      • Stillman B.
      • Speck C.
      ,
      • Remus D.
      • Beuron F.
      • Tolun G.
      • Griffith J.D.
      • Morris E.P.
      • Diffley J.F.
      ). To initiate replication, the preinitiation complex rearranges into a bidirectional minireplication factory (
      • Cuesta I.
      • Núñez-Ramírez R.
      • Scheres S.H.
      • Gai D.
      • Chen X.S.
      • Fanning E.
      • Carazo J.M.
      ,
      • Wessel R.
      • Schweizer J.
      • Stahl H.
      ,
      • Smelkova N.V.
      • Borowiec J.A.
      ,
      • Reese D.K.
      • Meinke G.
      • Kumar A.
      • Moine S.
      • Chen K.
      • Sudmeier J.L.
      • Bachovchin W.
      • Bohm A.
      • Bullock P.A.
      ,
      • Meinke G.
      • Phelan P.
      • Moine S.
      • Bochkareva E.
      • Bochkarev A.
      • Bullock P.A.
      • Bohm A.
      ). As Tag unwinds parental DNA, it interacts physically with two different surfaces of RPA and actively loads it onto the emerging template via a transient ternary complex with RPA/ssDNA, thereby coupling DNA unwinding with RPA deposition (
      • Weisshart K.
      • Taneja P.
      • Fanning E.
      ,
      • Arunkumar A.I.
      • Klimovich V.
      • Jiang X.
      • Ott R.D.
      • Mizoue L.
      • Fanning E.
      • Chazin W.J.
      ,
      • Jiang X.
      • Klimovich V.
      • Arunkumar A.I.
      • Hysinger E.B.
      • Wang Y.
      • Ott R.D.
      • Guler G.D.
      • Weiner B.
      • Chazin W.J.
      • Fanning E.
      ,
      • Bochkareva E.
      • Martynowski D.
      • Seitova A.
      • Bochkarev A.
      ). Tag also interacts physically with at least three subunits of pol-prim (
      • Dornreiter I.
      • Höss A.
      • Arthur A.K.
      • Fanning E.
      ,
      • Dornreiter I.
      • Erdile L.F.
      • Gilbert I.U.
      • von Winkler D.
      • Kelly T.J.
      • Fanning E.
      ,
      • Dornreiter I.
      • Copeland W.C.
      • Wang T.S.
      ,
      • Collins K.L.
      • Russo A.A.
      • Tseng B.Y.
      • Kelly T.J.
      ,
      • Huang S.G.
      • Weisshart K.
      • Gilbert I.
      • Fanning E.
      ,
      • Weisshart K.
      • Förster H.
      • Kremmer E.
      • Schlott B.
      • Grosse F.
      • Nasheuer H.P.
      ,
      • Ott R.D.
      • Rehfuess C.
      • Podust V.N.
      • Clark J.E.
      • Fanning E.
      ). These interactions led to a model of SV40 primosome activity in which Tag contacts with RPA/ssDNA remodel RPA into a weaker ssDNA-binding mode, transiently affording local access to the template (Fig. 1A). Tag can then load its associated pol-prim onto RNA-ssDNA in a molecular handoff reaction that enables primer synthesis (
      • Arunkumar A.I.
      • Klimovich V.
      • Jiang X.
      • Ott R.D.
      • Mizoue L.
      • Fanning E.
      • Chazin W.J.
      ,
      • Collins K.L.
      • Kelly T.J.
      ,
      • Melendy T.
      • Stillman B.
      ,
      • Yuzhakov A.
      • Kelman Z.
      • Hurwitz J.
      • O'Donnell M.
      ,
      • Fanning E.
      • Klimovich V.
      • Nager A.R.
      ). Thus, physical interactions among pol-prim, Tag, and RPA are proposed to contribute to primosome activity in the SV40 replisome.
      Figure thumbnail gr1
      FIGURE 1Tag 357–627 is sufficient to bind to pol-prim p68 1–107. A, a molecular handoff model for SV40 primosome activity on RPA-coated ssDNA. The four ssDNA-binding domains (A–D) of RPA (dark gray) occlude up to 30 nucleotides of ssDNA (straight line). Flexible linkers (wavy lines) join the N-terminal domain of RPA70 and the C-terminal domain of RPA32 to the RPA/ssDNA. Tag contacts with RPA32C and RPA70AB remodel it into a more compact, lower affinity ssDNA-binding mode and stabilize it as a ternary complex (
      • Arunkumar A.I.
      • Klimovich V.
      • Jiang X.
      • Ott R.D.
      • Mizoue L.
      • Fanning E.
      • Chazin W.J.
      ,
      • Jiang X.
      • Klimovich V.
      • Arunkumar A.I.
      • Hysinger E.B.
      • Wang Y.
      • Ott R.D.
      • Guler G.D.
      • Weiner B.
      • Chazin W.J.
      • Fanning E.
      ,
      • Fanning E.
      • Klimovich V.
      • Nager A.R.
      ), transiently exposing the template ssDNA. pol-prim (light gray) contacts the Tag helicase domains (HEL) through p68N (
      • Huang H.
      • Weiner B.E.
      • Zhang H.
      • Fuller B.E.
      • Gao Y.
      • Wile B.M.
      • Zhao K.
      • Arnett D.R.
      • Chazin W.J.
      • Fanning E.
      ), the N terminus of p180 DNA polymerase, and unknown surfaces of primase p58/p48 (PRI) (
      • Dornreiter I.
      • Copeland W.C.
      • Wang T.S.
      ,
      • Huang S.G.
      • Weisshart K.
      • Gilbert I.
      • Fanning E.
      ,
      • Weisshart K.
      • Förster H.
      • Kremmer E.
      • Schlott B.
      • Grosse F.
      • Nasheuer H.P.
      ). The ensemble of these interactions is proposed to position primase on the exposed template to synthesize an RNA primer (not shown). B, domain architecture of SV40 Tag. The DnaJ chaperone domain (
      • Kim H.Y.
      • Ahn B.Y.
      • Cho Y.
      ), SV40 OBD (
      • Luo X.
      • Sanford D.G.
      • Bullock P.A.
      • Bachovchin W.W.
      ), and helicase domain (
      • Li D.
      • Zhao R.
      • Lilyestrom W.
      • Gai D.
      • Zhang R.
      • DeCaprio J.A.
      • Fanning E.
      • Jochimiak A.
      • Szakonyi G.
      • Chen X.S.
      ,
      • Gai D.
      • Zhao R.
      • Li D.
      • Finkielstein C.V.
      • Chen X.S.
      ) are depicted. The structure of the host-range (HR) domain is not known (
      • Spence S.L.
      • Pipas J.M.
      ). C, GST-tagged Tag fragments 131–259 (lanes 2 and 3), 251–627 (lanes 4 and 5), 303–627 (lanes 6 and 7), or 357–627 (lanes 8 and 9) adsorbed to glutathione beads were incubated with increasing amounts of His-tagged p68 1–107 as indicated. Proteins bound to the beads were separated by SDS-PAGE and visualized by Western blotting with anti-His antibody (top) or anti-GST antibody (bottom). Glutathione beads lacking GST-Tag protein (lane 1) are shown as negative control. Lane 10 shows 200 ng of input p68 1–107.
      To gain greater insight into the operation of the SV40 primosome, we recently identified a previously unrecognized domain of the pol-prim p68 subunit (p68N) that docks on Tag, determined its solution structure, and identified the surface of p68N that docks on Tag (
      • Huang H.
      • Weiner B.E.
      • Zhang H.
      • Fuller B.E.
      • Gao Y.
      • Wile B.M.
      • Zhao K.
      • Arnett D.R.
      • Chazin W.J.
      • Fanning E.
      ). Structure-guided mutagenesis of p68N was used to confirm its Tag-docking surface. Substitutions in this surface that specifically reduced its affinity for Tag were then introduced into the intact pol-prim complex and shown to diminish SV40 primosome activity. The results demonstrated that p68-Tag docking is vital for primosome activity, even in the presence of p180 and primase docking on Tag, supporting a working model in which this network of contacts may position pol-prim to access the exposed template. This model implies the existence of a corresponding docking site for p68N on the surface of Tag. Localization of pol-prim-docking sites on Tag would provide new insight into the architecture of the primosome and coordination of its activity with that of the helicase.
      Here we report the identification of the predicted p68N-docking site on the C-terminal face of the Tag helicase domain, show that a Tag variant unable to bind to p68 retains binding to p180 and primase, and demonstrate the importance of p68-Tag interaction in primosome activity. In addition, we report that the ATPase/translocase activity (and hence helicase activity) of Tag is dispensable for primosome activity in vitro. Potential implications of our data for the overall architecture of the SV40 primosome and the coordination of priming with parental DNA unwinding are discussed.

      Acknowledgments

      We thank J. M. Pipas, X. S. Chen, R. D. Ott, W. J. Chazin, B. E. Weiner, G. D. Guler, X. Zhao, B. Zhou, W. C. Copeland, W. P. Dulaney, G. Sowd, E. Kremmer, and H. Zhang for advice, reagents, cooperation, and discussion.

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