Mechanism of DNA Interaction and Translocation by the Replicase of a Circular Rep-Encoding Single-Stranded DNA Virus

FIG 1. Architecture of PCV2 Rep-DNA. (A) Schematic of Rep with the endonuclease domain (ED, pastel yellow), oligomerization domain (OD, pastel red), and ATPase domain (AD, pastel cyan) labeled. The domain boundaries are identified by the amino acid numbers written at the top. (B) 2D class averages demonstrating different orientations of Rep visualized during cryo-EM imaging. The averages have been computationally color coded to identify the ED, OD, and AD using the color convention described above. (C) Orthogonal views of the Rep-ssDNA cryo-EM map showing top, side, and bottom views. The six subunits are color coded consistently throughout this paper. The OD is colored slightly darker than the AD. (D) Orthogonal views of the Rep-dsDNA cryo-EM map with orientations comparable to panel C. (E) Segmented subunits of the Rep-ssDNA cryo-EM map. The OD and AD are labeled on the left side. The OD of the six subunits were superposed to highlight the rigid body movement of the AD. Images in panels C to E generated using UCSF Chimera X.

FIG 2. Structure of PCV2 Rep-ssDNA. (A) Modeling of coordinates into the Rep-ssDNA cryo-EM map. (Left) Cryo-EM map with modeled regions colored in dark gray. (Center) Map shown as a chicken wire mesh with model as stick representation. (Right) Ninety-degree rotation of center orientation; note the arrow for direction of rotation. (B) Ribbon-stick-CPK representation of subunit B with helices in yellow, strands in cyan, and loops in gray. The OD and AD have been labeled. Functionally important residues are identified as thick sticks. ADP is shown as CPK. The N and C termini are indicated by blue and red circles. Image on right is 90° rotation; note the arrow for direction of rotation. Image generated using UCSF Chimera X. (C) Pipe-and-plank cartoon of hexamer with subunits labeled. Subunits colored as in Fig. 1. The cylinders in the center identify the axis of rotation for overlaying the AD of one subunit onto the subsequent subunit. The color of the cylinder identifies the subunit that is to be rotated. The stick model ssDNA can be seen in the center of the hexamer. Figure generated using UCSF Chimera.

FIG 3. Nucleotide binding pockets of Rep-ssDNA structure. Ribbon-and-stick cartoon showing the bound ADP to interfaces AB, BC, CD, and DE. Hydrogen bonds and electrostatic interactions are shown as dashed lines. The ADP is modeled as a gray stick model, its corresponding density as a transparent surface mesh, and the Mg2+ as a purple sphere. The surface representation on the top right of each panel highlights the nucleotide binding site (arrowhead) with respect to the hexamer. (A) AB subunit interface (ATP-like); (B) BC subunit interface (ATP-like); (C) CD interface (ADP); (D) DE interface (ADP); (E) EF interface (empty); (F) FA interface (empty). Images generated using UCSF Chimera and UCSF Chimera X.

FIG 4. Functional studies of PCV2 Rep. (A) The basal NTPase activity of Rep was measured using the ATP/NADH-coupled spectrophotometric assay under Michalis-Menten conditions. Rep can hydrolyze ATP, GTP, CTP, and UTP, with a preference for ATP. (B) Agarose gel of Rep demonstrating that it binds exclusively to DNA. Lane 1, 1-kbp ladder (Gold Biotechnology); lane 2, Rep at room temperature; lane 3, denatured Rep (dRep); lane 4, dRep treated with RNase I (2 h); lane 5, dRep treated with DNase I (1 h); lane 6, dRep treated with DNase I (2 h); lane 7, 12-nt ssDNA; lane 8, 44-nt ssDNA.

FIG 5. Rep DNA interaction. (A) (Left) Ribbon-stick cartoon of Rep in complex with ssDNA. The six subunits of Rep are colored and labeled according to Fig. 1. Amino acids Trp202 (pore loop 1) and Lys240 (pore loop 2) are shown as sticks. The interactions between Lys240 and ssDNA are shown as dashed lines. (Right) Stick cartoon of ssDNA and Rep rotated by 90°. The 5′-PO4 and 3′-OH of ssDNA have been labeled, and the spiral staircase arrangement of Trp202 and Lys240 is demonstrated. (B) (Left) Ribbon-stick cartoon of Rep interacting with dsDNA. The interactions described for Rep-ssDNA persist. Image focuses on the interaction between Rep and the second strand of DNA—identical orientation as shown in panel A. (Right) Ninety-degree rotation of model on left. The side chains for Trp202 and Lys240 are shown. The interactions between Arg199 and the second strand of DNA are shown as dashed blue lines.

FIG 6. Translocation of ssDNA by Rep. The six images on top demonstrate the translocation of each Rep AD. The six images on the bottom identify the location of the AD within the staircase arrangement. Rep subunits move from one position to the next to translocate ssDNA (6 → 1, 1 → 2, 2 → 3, 3 → 4, 4 → 5, and 5 → 6). Subunits A to F are color coded according to Fig. 1. The dsDNA fork is shown at the top, with the displaced ssDNA moving to the left. dsDNA is shown at the bottom of Rep, where its 3′-OH serves as the DNA elongation site during replication. Attention is given to the subunits A (pastel magenta), F (pastel red), and E (pastel yellow). The Arabic numbers identify the position of each subunit on the staircase. Binding of ATP to the nucleotide binding site of the subunit at position 6 (bottom of staircase) promotes its translocation to position 1 (top of the staircase). Consequently, each subunit moves to the next position in the staircase. Binding of ATP to the 6-1 interface and release of ADP from the 4-5 interface are shown at the bottom using filled and unfilled arrows. Each step translocates Rep by ∼3.5 Å toward the dsDNA fork.