Replisome Assembly at oriC, the Replication Origin of E Replisome Assembly at oriC, the Replication Origin of E. coli, Reveals an Explanation for Initiation Sites outside an Origin  Linhua Fang, Megan J Davey, Mike O'Donnell  Molecular Cell  Volume 4, Issue 4, Pages 541-553 (October 1999) DOI: 10.1016/S1097-2765(00)80205-1

Figure 5 Mapping of RNA/DNA Junctions of Initial Start Sites at oriC Primer extension using 32P-labeled primers was used to determine the location of RNA/DNA junctions on replicated pUC18 oriC as described under Experimental Procedures. (A) Autoradiogram of a sequencing gel of 32P-primer extension products that map RNA/DNA junctions on the bottom strand. (B) Autoradiogram of 32P-primer extension products on the top strand. The positions of the 13-mer repeats (L, M, and R) and AT-rich region in oriC are illustrated on the side of the gels. (C) Summary of initiation sites using 96 nM primase. Vertical line heights reflect relative intensities in the sequencing gels. Horizontal arrows indicate direction of DNA synthesis. The vertical arrow, at position 392358 9 (+1), indicates the left boundary of oriC. Numbers reflect the position in the E. coli genome. The number within parentheses indicates the position in the minimal oriC sequence (Kornberg and Baker 1992). (D) Bidirectional replication was confirmed using pBROTB (shown in upper panel) as described in Experimental Procedures. The 2 and 3 kb fragments between terB sites and oriC are indicated on the plasmid map. An autoradiogram of a denaturing agarose gel of the replication products (lower panel) with increasing amounts of DnaB (as indicated) is shown. Quantitation of the 2 and 3 kb bands using a phosphorimager indicates equal amounts of each in every lane (taking into account differences in intensity due to their different sizes). Molecular Cell 1999 4, 541-553DOI: (10.1016/S1097-2765(00)80205-1)

Figure 1 Assembly of Proteins at the Origin Five different experiments, differing only in which radioactive-labeled protein was added in place of the unlabeled protein, were performed as described in Experimental Procedures. Proteins were assembled onto plasmid DNA either containing oriC (closed circles), or lacking it (open circles) followed by gel filtration. Radiolabeled proteins were located in column fractions by liquid scintillation and quantitated from their known specific activity: (A) 32P-DnaB, (B) 3H-DnaC, (C) 3H-Pol III*, (D) 3H-β, and (E) 32P-primase. The diagram at the top summarizes the results. Two hexamers of DnaB are shown as encircling ssDNA. The two ring-shaped β clamps are each shown as a torus encircling a primed site. Each Pol III* contains two core polymerases and one γ complex clamp loader (not shown). Molecular Cell 1999 4, 541-553DOI: (10.1016/S1097-2765(00)80205-1)

Figure 2 Characterization of DnaB ATP Site Mutants Assays were performed as described under Experimental Procedures. In each assay, all components were kept the same except for the type of DnaB used: DnaBPK (circles), DnaBPKK236R (diamonds), DnaBPKK236A (triangles), or no DnaB ([D] only, squares). (A) ATP binding. (B) ATPase activity using M13mp18 ssDNA as a DNA effector. (C) Oligonucleotide displacement from M13mp18 ssDNA. (D) General priming activity testing the ability of different DnaB proteins to support general priming by primase on M13mp18 ssDNA. (E) oriC replication assays. Molecular Cell 1999 4, 541-553DOI: (10.1016/S1097-2765(00)80205-1)

Figure 3 Two DnaB Hexamers Assemble onto oriC in the Absence of Helicase Action DnaB assembly experiments are described in Experimental Procedures. (A) Assembly of DnaBPK (left panel) or DnaBPKK236A (right panel) onto M13mp18 ssDNA in the presence (triangles) or absence (circles) of DnaC. (B) 32P-labeled DnaBPKK236A and 3H-DnaC were incubated with plasmid DNA along with DnaA protein, HU, and SSB. Assembly of DnaBPKK236A, left panel; 3HDnaC (right panel); pUC18 oriC, triangles; pUC18, circles. Molecular Cell 1999 4, 541-553DOI: (10.1016/S1097-2765(00)80205-1)

Figure 4 Helicase Action Is Needed for Priming and Replisome Assembly (A) Assembly of Pol III* (left panel) and β (right panel) on pUC18 oriC was performed using either DnaBPK (closed circles) or DnaBPKK236A (open circles). (B) RNA synthesis on pUC18 oriC was performed using either DnaBPK (closed circles) or DnaBPKK236A (open circles). Molecular Cell 1999 4, 541-553DOI: (10.1016/S1097-2765(00)80205-1)

Figure 6 Footprint Analysis of DnaB on oriC Using KMnO4 (A) KMnO4 modification experiments were performed as described under Experimental Procedures. In brief, pUC18 oriC was incubated with DnaA and/or DnaBPKK236A plus DnaC, then treated with KMnO4, followed by location of the modified residues by primer extension analysis in a sequencing gel. The left gel is the upper strand analysis, and the lower strand is shown to the right. All reactions except lane 1 contained 0.46 μg DnaA. Lane 1, 0.35 μg DnaBPKK236A; lane 2, no DnaBPKK236A; lane 3, 0.18 μg DnaBPKK236A; and lane 4, 0.35 μg DnaBPKK236A. DnaC was added at an equimolar ratio to DnaBPKK236A (monomer to monomer). The diagram between the gels shows ssDNA in the DnaA-induced open complex (middle diagram) compared to the DnaBC-induced expanded melted region of the origin to include the L 13-mer and AT-rich region (bottom diagram). The direction of helicase movement is indicated by the arrows. (B) Scale representation of DnaB on a 55 nt bubble. The dimensions of DnaB are based on those from electron microscope studies. Molecular Cell 1999 4, 541-553DOI: (10.1016/S1097-2765(00)80205-1)

Figure 7 Stages in Assembly of Two Opposed Replication Forks at oriC (A) Stages in replisome assembly. Stage I, two DnaB hexamers are assembled onto the DnaA-activated open complex through the action of DnaC. Stage II, DnaB helicases pass each other creating ssDNA for primase action. The passing action ensures that the region between the helicases is melted and remains so upon being coated with SSB. Primase must interact with DnaB to initiate RNA synthesis resulting in RNA primers in cis to DnaB. Stage III, two replicases assemble onto the two primed sites. Stage IV, the two molecules of Pol III holoenzyme extend DNA opposite the motion of DnaB assembled on the same strand. Hence, the polymerases pass one another to reach the DnaB helicases on the opposite strand, which move in the same direction as DNA polymerization. (B) The factory model for replication indicates that the polymerases remain fixed while the DNA moves. Molecular Cell 1999 4, 541-553DOI: (10.1016/S1097-2765(00)80205-1)