The Control Mechanism for Lagging Strand Polymerase Recycling during Bacteriophage T4 DNA Replication  Jingsong Yang, Scott W. Nelson, Stephen J. Benkovic  Molecular Cell  Volume 21, Issue 2, Pages 153-164 (January 2006) DOI: 10.1016/j.molcel.2005.11.029 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Effect of dCTP Concentrations on Replisomal Replication Using Minicircle Substrate (A) The 70 bp minicircle substrate. The leading strand template lacks guanine residues, enabling selective control and monitoring of leading and lagging strand synthesis. The boxes indicate priming sites that are located 35 bp apart. (B) Uncoupling of the leading and lagging strand polymerases by selectively slowing down the lagging strand polymerase at reduced [dCTP] (2.5 μM). Reactions were carried out for 2 min under the standard conditions found in the Experimental Procedures to ensure complete formation of replication forks and followed by a 20-fold dilution into buffer containing [8-3H]dGTP and [α-32P]dCTP and all reaction components except for the minicircle substrate and dCTP. (C) Effect of [dCTP] on the size of the Okazaki fragments. Lagging strand synthesis was monitored in the presence of [α-32P]dCTP on the minicircle substrate at decreasing dCTP concentrations. (D) The peak of the Okazaki fragment distribution shown in (C) plotted against [dCTP]. Molecular Cell 2006 21, 153-164DOI: (10.1016/j.molcel.2005.11.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Gap Filling by the pfuTurbo and Deep Vent Polymerases on the Lagging Strand Products Synthesized at Various dCTP Concentrations (A) Standard minicircle replication reactions were carried out at 50, 10, and 2.5 μM dCTP in the presence of [α-32P]dCTP to monitor the lagging strand synthesis. The reaction products were purified as described in the Experimental Procedures and extended by pfuTurbo or Deep Vent polymerases. (B) Schematic showing the presence of ssDNA gaps on the lagging strand template and their subsequent filling in by thermophilic polymerases. Molecular Cell 2006 21, 153-164DOI: (10.1016/j.molcel.2005.11.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Replication Reactions on pONE_nick Substrate (A) The pONE_nick substrate. The primase recognition and nick sites are shown by GTT and 3′OH, respectively. (B) Replication reactions on pONE_nick were carried out as described in the Experimental Procedures in the presence of all four rNTPs or ATP/CTP only. Lanes 1–3 represent time points of 5, 10, and 15 min, respectively. (C) The size distribution of the replication products in the presence of ATP/CTP. Eight individual peaks can be detected at 5 (light gray), 10 (dark gray), and 15 min (black). (D) The peak distribution over time. The numbered peaks from (B) are plotted in normalized Okazaki fragment intensity. The intensity of peak one is 41% of the total peak intensities, which represents the value for primer utilization with this substrate. Molecular Cell 2006 21, 153-164DOI: (10.1016/j.molcel.2005.11.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Replication Reactions on pONE_nick Substrate with Varying Amounts of CTP (A) Rolling circle replication with pONE_nick substrate in the presence of ATP and varying amounts of CTP. Lanes 1–6 contain 0, 2.5, 5, 10, 20, and 40 μM CTP. The product distributions of lanes 1 (light gray), 2 (black), and 3 (dark gray) are shown below the gel. (B) Replication reactions on pONE_nick were initiated in the presence of 2.5 μM CTP and allowed to proceed for 7 min before a 10-fold dilution into buffer containing [α-32P]dCTP and all reaction components except for the pGEM substrate. Additional CTP was added after 2 min to a final concentration of 100 μM. Lanes 1–3 are 0.5, 1, and 1.5 min after addition of [α-32P]dCTP, respectively. Lanes 4–6 are 0.5, 1, and 1.5 min after the addition of enough CTP to bring the final concentration to 100 μM, respectively. The product distributions of lanes 3 (gray) and 4 (black) are shown below the gel. The arrow denotes the appearance of the 1.1 kb Okazaki fragment after the increase in CTP levels. Molecular Cell 2006 21, 153-164DOI: (10.1016/j.molcel.2005.11.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Effect of Clamp and Clamp Loader Concentrations on Primer Utilization and the Size of Okazaki Fragments (A and B) Replication reactions were carried out as described in the Experimental Procedures. [α-32P]CTP was included in all reactions to label the RNA primers synthesized. Concentrations of clamp and clamp loader were 0.025, 0.1, 0.25, 0.5, and 1 μM ([A], lanes 1–5) and 0.0625, 0.125, 0.25, 0.5, and 1 μM ([B], lanes 1–5) for replication reactions on minicircle and pGEM_nick, respectively. (C) Decrease in the size of Okazaki fragments at increasing [clamp/clamp loader] concentrations. Replication reactions were carried out in the presence of [α-32P]dCTP on the pGEM_nick substrate. Molecular Cell 2006 21, 153-164DOI: (10.1016/j.molcel.2005.11.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Okazaki Fragment Size Distribution Determined by Markov Chain Monte Carlo Simulation The simulated data were generated as described in the Experimental Procedures. (A) Raw output from the computer simulation. The solid line and dotted lines represent the data from stochastic and deterministic simulations, respectively. (B) Comparison between the stochastically simulated and experimentally determined EM data. The simulated (black bars) and EM (gray bars) data are presented as histograms with a bin width of 300 bp. The EM data were compiled from Table I of Chastain et al. (2000). Molecular Cell 2006 21, 153-164DOI: (10.1016/j.molcel.2005.11.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Proposed Model for the Primer Handoff Process of T4 Lagging Strand Synthesis (A) During the replication phase of the Okazaki fragment synthesis, an RNA primer (shown in red) is synthesized by the primase. (B) A clamp protein is recruited from solution and loaded onto the newly synthesized RNA primer assisted by the clamp loader. (C) Loading of the clamp subsequently triggers the dissociation of the lagging polymerase. (D) Lagging strand polymerase is recycled onto the new RNA primer for the assembly of the holoenzyme complex to initiate the next Okazaki fragment synthesis. Molecular Cell 2006 21, 153-164DOI: (10.1016/j.molcel.2005.11.029) Copyright © 2006 Elsevier Inc. Terms and Conditions