Volume 25, Issue 1, Pages 157-166 (January 2017) Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome  Jamie R. Wallen, Hao Zhang, Caroline Weis, Weidong Cui, Brittni M. Foster, Chris M.W. Ho, Michal Hammel, John A. Tainer, Michael L. Gross, Tom Ellenberger  Structure  Volume 25, Issue 1, Pages 157-166 (January 2017) DOI: 10.1016/j.str.2016.11.019 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2017 25, 157-166DOI: (10.1016/j.str.2016.11.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 A Three-Polymerase Replisome View looking at the C-terminal face of the gp4 heptameric ring (colored gray; left). An HTH motif (residues 366–387, colored orange) of gp4 lies on the outside surface of the ring and contacts all three polymerases. Pols A, B, and C are colored green, magenta, and cyan, respectively. A 90° rotation of the structure (right) illustrates that the primase domains of gp4 (colored white) do not contact the polymerases. Structure 2017 25, 157-166DOI: (10.1016/j.str.2016.11.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 The Gp4 C-Terminal Tail Inserts into a Pocket Near the Front Basic Patch of Gp5/trx (A) A 2Fo-Fc map (blue), contoured at 1σ, is shown for the C-terminal amino acid (Y547) of gp4 included in the crystallographic model. An Fo-Fc map contoured at 2.5σ (green) reveals additional electron density leading from the C-terminal tail of gp4 (colored yellow) to a hydrophobic pocket adjacent to the front basic patch of Pol B. The electrostatic surface potential of Pol B is shown with residues K589 and R590 from the front basic patch labeled. An analogous patch of density is observed near the front basic patch of Pol A (data not shown). (B) The C-terminal tail of gp4 (light brown) bypasses Pol C (colored by electrostatic surface potential). The front basic patch of Pol C is partially occluded by its interaction with gp4 so that the tail of gp4 does not engage in the binding interaction observed for Pol B and Pol A. 2Fo-Fc electron density (blue) contoured at 1σ is shown for the gp4 tail. Structure 2017 25, 157-166DOI: (10.1016/j.str.2016.11.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Pol B Makes Extensive Interactions with Gp4 while Pol C Binds in a Skewed Orientation that Precludes Interactions with the Gp4 Tail (A) Pol B contacts the gp4 ring on a convoluted interface that spans the fingers, palm, and exonuclease subdomains of Pol B and two subunits of gp4. A ribbon model of Pol B is colored for residues within 8 Å of the gp4 surface according to the Kyte-Doolittle hydropathy values (Kyte and Doolittle, 1982) ranging from polar residues (purple) to hydrophobic residues (yellow). The solvent accessible surface of gp4 is similarly colored according to hydrophobicity on a gray or white background depicting adjacent gp4 subunits. Pol B helices L, P, and Q circumscribe a hydrophobic pocket that accepts F566 from the tail of gp4. Residues from the front basic patch of Pol B, located in the connecting loop between helices P and Q, contact acidic residues (colored purple) in the gp4 tail. Another prominent interaction is made by the HTH motif of gp4, which inserts in a groove located between Pol B helices R, G, and G1. Gp4 tail residues 550–566 are disordered in the crystal structure and were modeled here then subjected to MD simulation, which revealed a very stable interaction of F566 with Pol B (Table S1). (B) Pol C (dark gray) is shown in the foreground bound next to Pol B on the surface of gp4. Many of the same residues of Pol C and Pol B make contact with gp4, but the rotated orientation of Pol C creates different sets of interactions with less buried surface area featuring predominately polar residues (purple) and fewer hydrophobic residues (yellow). Residues within 8 Å of the binding sites for Pol B and Pol C are colored according to the Kyte-Doolittle hydropathy values on a scale ranging from polar residues (purple) to hydrophobic residues (yellow), as for (A). Another important consequence of the alternative binding orientation of Pol C is that the gp4 tail-binding pocket comprising helices L, P, and Q of Pol C is occluded, preventing interactions with the tail, which are prominent for Pol A and Pol B (cf. Figure 2B). Without these tail interactions, the Pol C interface supports fewer hydrogen-bonding interactions with gp4 (Table S2) in comparison with the Pol B interface (Table S1). Structure 2017 25, 157-166DOI: (10.1016/j.str.2016.11.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Native MS Reveals Different T7 Replisome Assembly States (A) A titration of gp5/trx against a fixed concentration of gp4 shows the assembled, ring-shaped gp4 helicase binding to one, two, and three copies of the polymerase. (B) At low collision voltage, complexes containing two copies of gp5/trx are of comparable abundance with assemblies containing three copies of gp5/trx. The three-copy gp5/trx complexes become less abundant upon collisional activation at higher voltages, indicating they are less stable than those with two copies of gp5/trx. The peak shapes are broadened at lower collision voltages, strongly suggesting incomplete desolvation and making it difficult to deduce quantitatively the relative binding affinity of the third gp5/trx with the gp4 hexamer and heptamer. Structure 2017 25, 157-166DOI: (10.1016/j.str.2016.11.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 SAXS Reveals a Dynamic Structure of the T7 Replisome (A) Normalized P(r) functions for gp4Δprimase alone (green) and a 1:1 complex of gp4Δprimase hexamer and gp5/trx either in the absence (black) or presence (blue) of a primer/template DNA. Calculated P(r) functions are shown for structures of the gp4Δprimase hexamer bound to two copies of DNA polymerase oriented as observed for Pol A and Pol B in the structure (gray dots) and bound to three polymerase molecules (Pols A, B, and C) as observed in the crystal structure (light gray dots). The P(r) functions are normalized based on the molecular volume of the complexes determined by SAXS, as indicated in (D). (B) Raw scattering profiles of the 1:1 complex in the absence of a primer/template DNA (black) and a theoretical scattering profile obtained from our crystal structure (magenta) reveals that the structure in solution differs from a model based on the interaction of Pol B with gp4 in the crystallized replisome. An ab initio docking model generated by FoXSDock (red) shows a better fit (χ = 2.7) to the experimental data than the Pol B-gp4Δprimase complex model (χ = 7.2). Inset: Guinier plot for experimental data with a limit of (q × Rg) < 1.6. In (B) and (C) the orientation of Pol B in the crystal structure is shown as a transparent surface (magenta), and the residuals between the experimental SAXS data and the models are shown below the raw scattering profiles. (C) The scattering profile of the 1:1 complex with a primer/template DNA (blue) reveals a unique structure in solution compared with the no-DNA sample and the crystal structure. A FoXSDock model (cyan, χ = 3.5) shows the polymerase on the outside of the ring, which could be generated by a rotation of the polymerase as observed in our crystal structure. An ensemble model calculated by FoXS (orange trace) predicts that our experimental data are best described by a mixture of the FoXSDock model (57%) and the Pol B-gp4Δprimase model (43%). This ensemble of two models improves the fit (χ = 2.6) to the high q region of the experimental scattering data. Inset: Guinier plot for experimental data with a limit of (q × Rg) < 1.6. (D) SAXS-based structural parameters calculated from the experimental data shown in (A) to (C). MW, molecular weight. Structure 2017 25, 157-166DOI: (10.1016/j.str.2016.11.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Model of DNA Binding in the T7 Replisome Docking model of the T7 replisome complex bound to a replication fork DNA. The leading and lagging strands are colored slate blue and orange, respectively. As single-stranded DNA passes through the central channel of the helicase, the displaced leading strand is diverted to Pol A for leading-strand synthesis. The modeled lagging strand DNA is redirected by a replication loop to interact with Pol B. In this way, the crystal structure of the T7 replisome is suggestive of a simple mechanism for coupled replication of two antiparallel DNA strands. Structure 2017 25, 157-166DOI: (10.1016/j.str.2016.11.019) Copyright © 2016 Elsevier Ltd Terms and Conditions