Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4 Davis Jose, Steven E. Weitzel, Debra Jing, and Peter H. von Hippel1 Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403 Contributed by Peter H. von Hippel, July 9, 2012 (sent for review May 13, 2011) Physical biochemical techniques are used to establish the structure, rings of identical subunits and interact with one or more primase subunit stoichiometry, and assembly pathway of the primosome subunits to form functional primosomes (5–7). Thus, the T4 DNA complex of the bacteriophage T4 DNA replication system. Analyti- replication system and its constituent subassemblies comprise the cal ultracentrifugation and fluorescence anisotropy methods show simplest model system for studying many aspects of DNA repli- that the functional T4 primosome consists of six gp41 helicase sub- cation in higher organisms (8). Furthermore, each of the three units that assemble into a hexagon, driven by the binding of six functional protein subassemblies can be loaded onto an appropri- NTPs (or six nonhydrolyzable GTPγS analogues) that are located ate DNA construct in vitro, permitting their mechanisms to be at and stabilize the intersubunit interfaces, together with a single studied separately. DNA helicase activity is required to unwind tightly bound gp61 primase subunit. Assembling the components the genomic DNA at the replication fork and to provide access of the primosome onto a model DNA replication fork is a multistep for the DNA polymerases to their respective templates. This heli- process, but equilibrium cannot be reached along all mixing path- case function must be integrated with that of the primase, which ways. Producing a functional complex requires that the helicase serves as a low processivity RNA polymerase that carries out tem- hexamer be assembled in the presence of the DNA replication fork plate-directed synthesis of RNA (5-mer) primers at approxi- construct prior to the addition of the primase to avoid the forma- mately 1-kb intervals along the lagging strand DNA template. tion of metastable DNA-protein aggregates. The gp41 helicase hex- This functional integration of the helicase and the primase com- amer binds weakly to fork DNA in the absence of primase, but ponents of the T4 system alters both activities within the replica- forms a much more stable primosome complex that expresses full tion complex, and these interactions, together with the assembly and functional helicase (and primase) activities when bound to a pathway and subunit composition of the T4 primosome, form the gp61 primase subunit at a helicase:primase subunit ratio of 6∶1. main focus of this paper. The presence of additional primase subunits does not change the Biophysical studies of T4 primosome complexes (6, 7, 9, 10) molecular mass or helicase activity of the primosome, but signifi- have provided some information about their subunit stoichi- cantly inhibits its primase activity. We develop both an assembly ometry, as well as insight into how these components work in pathway and a minimal mechanistic model for the structure and conjunction with one another. However, two different sets of function of the T4 primosome that are likely to be relevant to observations have come to different conclusions about the stoi- the assembly and function of the replication primosome subassem- chiometry (and thus the mechanism) of the T4 primosome. In blies of higher organisms as well. early work gel shift and protein–protein chemical cross-linking methodologies had shown that the gp61 primase subunit binds DNA–protein complexes ∣ macromolecular machines ∣ to the gp41 helicase hexamer at an approximately 1∶6 subunit duplex DNA unwinding ∣ replication complex assembly stoichiometry (5, 7). In contrast, Benkovic and coworkers have interpreted more recent and indirect fluorescence anisotropy he DNA replication system of bacteriophage T4 contains and scanning calorimetry experiments (10–12) to argue that the Teight different types of protein subunits, several present in primase subunits may interact with gp41 in an approximately 6∶6 multiple copies. Subsets of these components form three stable subunit stoichiometry. This difference is important because in a and functional protein complexes that can be assembled onto primosome containing one primase and six helicase subunits the a model DNA replication fork in vitro to form an integrated primase must be shared, which makes very different functional T4 DNA replication complex that is capable of unwinding the and mechanistic demands on both helicase and primase mechan- parental DNA duplex and synthesizing new viral DNA with isms than does a 6∶6 ratio, as is found in the hexameric helicase of essentially in vivo rates and fidelity (1, 2). These replication sub- bacteriophage T7, where every helicase subunit carries its own assemblies are: (i) the leading- and lagging-strand replication primase domain (13). The replication complex of T7 differs in polymerases that catalyze the template-directed copying of the many important aspects from that of T4 or the replication com- two parental-strands of the DNA genome at each cell division; plexes of higher organisms. (ii) the clamp-clamp loader complex that controls the processivity Given the importance of the bacteriophage T4 replication of the replication process by linking the polymerases to their complex as a model system, we have undertaken additional and respective template strands and also regulates the release and re- careful studies by several methodologies to further examine the cycling of the lagging strand polymerase following the completion issues of subunit stoichiometry and assembly of the T4 primo- of each Okazaki fragment; and (iii) the helicase-primase (primo- some. The results increase our understanding of the molecular some) complex that unwinds the double-stranded genome ahead of the replication fork in its capacity as a helicase, while also per- Author contributions: D. Jose and P.H.v.H. designed research; D. Jose, S.E.W. and D. Jing forming template-directed synthesis of the RNA primers that re- performed research; D. Jose, S.E.W., D. Jing and P.H.v.H. analyzed data; and D. Jose and initiate discontinuous lagging-strand DNA synthesis after each P.H.v.H. wrote the paper. Okazaki fragment has been completed. The authors declare no conflict of interest. The subunit components and stoichiometries of the replication 1To whom correspondence should be addressed. E-mail: [email protected]. complexes of higher organisms closely resemble those of T4 This article contains supporting information online at www.pnas.org/lookup/suppl/ (3, 4). Their helicases, in the presence of NTP, all form hexameric doi:10.1073/pnas.1210040109/-/DCSupplemental. 13596–13601 ∣ PNAS ∣ August 21, 2012 ∣ vol. 109 ∣ no. 34 www.pnas.org/cgi/doi/10.1073/pnas.1210040109 Downloaded by guest on September 24, 2021 interactions that regulate primosome assembly and its function as a helicase and a primase, as well as providing insight into inter- actions between the primosome and the other subcomplexes of the T4 DNA replication system and, by extension, our under- standing of helicase-primase complexes in higher organisms. Results Assembly of the T4 helicase hexamer depends on the concentra- tions of gp41 subunits and NTP ligands. Analytical ultracentrifu- gation was used to characterize the equilibria involved in the assembly of the T4 gp41 helicase hexamer. Sedimentation velo- city profiles of gp41 at a subunit concentration of 1.5 μM in the absence of NTP ligands showed only one significant peak with an s20;w of approximately 4.5 S (Fig. 1A; see also Fig. S1). Assuming average hydration, this is close to the sedimentation coefficient expected for these relatively spherical gp41 subunits (14), and confirms earlier findings that gp41 protein exists in solution pri- marily as a monomer at these protein concentrations in the ab- sence of NTP (6). We confirmed also that the addition of GTPγS Fig. 1. Oligomerization of T4 helicase (gp41) subunits as a function of gp41 (a nonhydrolyzable analogue of GTP) to a solution of gp41 mono- and GTPγS concentrations. Sedimentation velocity profiles [(c)s versus s20;w μ γ mers at this concentration drives this replication helicase into a distribution plots] at: (A)1.5 M gp41 (monomers) with no GTP S; (B) 3 μM gp41 and 5 μM GTPγS; (C)3μM gp41 and 30 μM GTPγS; and (D) stable hexameric state. We note that two additional components μ μ γ s γ 3 M gp41 and 60 M GTP S. The approximately 4.5 S peak corresponds appeared in the c(s) versus 20;w distribution plots as the GTP S to gp41 monomers and the peak at approximately 10.5 S to gp41 hexamers. B C concentration was increased (Fig. 1 and ). These components The peaks at intervening s20;w values represent intermediate oligomers of fall at s20;w values between that of the monomer (approximately gp41 subunits and show a progressive shift into hexamers as the concentra- 4.5 S) and the approximately 10.5 S peak that corresponds to the tion of GTPγS is increased. (Additional plots showing complete sedimentation s sedimentation of the hexamer form that dominates at high profiles and residual values for the (c)s versus 20;w distribution plots of B and GTPγS concentrations (Fig. 1 C and D and Fig. S1F).* We sug- D are presented in Fig. S1.) gest that these intermediate peaks represent hexamer assembly intermediates, with the approximately 6 S peak corresponding cence anisotropy