Why is the leading strand synthesized continuously during DNA replication but the lagging strand is synthesized as Okazaki fragments This is because?

Size of the Okazaki Fragments Decreases with a Selective Decrease in the Rate of Lagging Strand Polymerization

To distinguish between the collision and the signaling mechanisms, we first studied the change in Okazaki fragment size caused by a selective decrease in the polymerization rate of the lagging strand polymerase. If the collision model dictates lagging strand polymerase recycling, slowing down the rate of lagging strand, but not leading strand, synthesis would lead to a progressive imbalance in the lengths of the respective template strands. Consequently, longer lagging strand DNA template loops will be generated that would result in the formation of longer Okazaki fragments. However, if the signaling mechanism is in effect under conditions where the frequency of the signal exhibited for lagging strand polymerase recycling is not affected, slowing down the lagging holoenzyme will result in the formation of shorter Okazaki fragments.

Because the minicircle substrate allows for the selective incorporation of dCTP into the lagging strand [Figure 1A], we utilized this substrate in the replication reactions to perturb only the rate of lagging strand synthesis. A series of reactions were carried out at decreasing dCTP concentrations. A control experiment demonstrates that the leading strand polymerase rate is unaffected by a reduction in the concentration of dCTP from 50 to 2.5 μM, whereas the rate of the lagging strand polymerase is reduced ∼3-fold [Figure 1B]. Because the Km for nucleotide was estimated to be around 10 μM [

], a drop of [dCTP] from 5 Km to 1/4 Km is expected to decrease the lagging strand polymerization rate by ∼4-fold, which is near the observed value. This result also confirms that leading and lagging strand synthesis can become uncoupled under certain conditions [

]. Moreover, the priming activity of primase, as monitored in a simple priming assay [

] was unchanged over a dCTP concentration range of 0.0625–50 μM, indicating that dCTP does not compete with CTP during primer synthesis [data not shown]. As shown in Figures 1C and 1D, lowering dCTP concentrations from 80 to 2.5 μM results in a decrease in Okazaki fragment size from 1.15 to 0.30 kb [Okazaki fragment size values reflect the peak of the distribution, i.e., the most frequent Okazaki fragment length]. This result is consistent with the signaling, rather than the collision, model being the major mechanism for lagging polymerase recycling.

Figure 1Effect of dCTP Concentrations on Replisomal Replication Using Minicircle Substrate

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[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].

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One possible alternative explanation for the formation of shorter Okazaki fragments is that the processivity of the lagging polymerase may be reduced at lower [dCTP], which would lead to premature termination of Okazaki fragment synthesis. To account for the size of the observed Okazaki fragments, a short dissociation half-life of ∼0.07 min for the lagging polymerase is required. To rule this out, we directly determined the half-life of the lagging strand polymerase at low [dCTP] by the dilution method described previously [

]. The measured koff is 0.14 min−1 at 2.5 μM dCTP [Figure S2 available in the Supplemental Data with this article online], which corresponds to a half-life of ∼7 min and is not significantly changed from the koff observed at 50 μM dCTP [

]. This value is 100-fold greater than the required 0.07 min half-life and indicates that an active mechanism that interrupts the normal processivity must be responsible for polymerase recycling at both normal and low [dCTP].

Another possibility, although less likely, is that a certain fraction of the replication forks may be doing leading strand synthesis only at low [dCTP] and generating long ssDNA tails. The shorter lagging strand product may come from the nonspecific primer extension reactions on these ssDNA tails. To exclude this possibility, we generated a minicircle substrate with ssDNA tails of up to 30 kb in length. This substrate was added to a solution containing all T4 components needed for in vitro replication except dGTP. Omission of dGTP ensured that no leading strand replication took place. Therefore, any product observed should come from nonspecific primer extension reactions. Even though primers were synthesized, we did not observe significant DNA product formation [Figure S3], indicating that nonspecific extension of primers outside the context of the replisome is very inefficient under our experimental conditions.

ssDNA Gaps between Okazaki Fragments Become More Prominent When the Rate of the Lagging Strand Polymerase Is Reduced as Compared to the Leading Strand Polymerase

If the shorter Okazaki fragments formed in the previous experiment were due to release of the lagging polymerase from DNA before encountering the end of the previous Okazaki fragment, one would expect to observe the formation of ssDNA gaps on the lagging strand. We reasoned that the gaps thus formed could be filled by polymerases catalyzing the extension of the 3′ primer strand. Thermophilic polymerases [Deep Vent and pfuTurbo] were used in these experiments. One advantage of using thermophilic polymerases is that high reaction temperature helps eliminate possible secondary structures within ssDNA regions, allowing the extension reaction to progress smoothly.

The extension reactions were performed as described in the Experimental Procedures. As shown in Figure 2, the Okazaki fragments generated at 2.5 μM dCTP were extended by pfuTurbo polymerase from 0.38 to 1.2 kb, and those generated at 10 μM dCTP were extended from 0.55 to 1.2 kb. Also noteworthy is a small change in the size from 0.85 to 1.2 kb of those formed at optimal [dCTP] [50 μM], a condition where the rates of polymerase at both strands are the same, i.e., the two holoeznymes are coupled. These results thus demonstrate the presence of large ssDNA gaps formed on the lagging strand at lower [dCTP] and smaller gaps formed at normal [dCTP], which is inconsistent with the collision model that predicts no gaps in lagging strand synthesis.

Figure 2Gap Filling by the pfuTurbo and Deep Vent Polymerases on the Lagging Strand Products Synthesized at Various dCTP Concentrations

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[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.

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We noted that the extension products in the Deep Vent reaction were slightly longer than those in the pfuTurbo reaction [1.4 and 1.2 kb, respectively]. Unlike pfuTurbo, DeepVent polymerase is able to perform strand-displacement synthesis. As a result, when reaching the end of the previous Okazaki fragment, a certain fraction of the Deep Vent polymerase was able to continue the extension reaction, resulting in the formation of longer extension product. This pattern of product formation is consistent with gapped lagging strand products acting as primers.

The presence of ssDNA gaps indicates that solution gp43 polymerase has not completely filled in these gaps under our experimental conditions. Although the concentration of polymerase used in our assays is nearly stoichometric with DNA [leading and lagging strand], it is assumed that a certain amount of polymerase will be available for the ssDNA gap filling reaction. The reason for the absence of gap filling is currently unknown. However, we believe that gp43 polymerase activity is likely inhibited by the high concentration of gp59 in the replication reactions [

Xi et al., 2005

• Xi J.
• Zhuang Z.
• Zhang Z.
• Selzer T.
• Spiering M.M.
• Hammes G.G.
• Benkovic S.J.

Interaction between the T4 helicase-loading protein [gp59] and the DNA polymerase [gp43]: a locking mechanism to delay replication during replisome assembly.

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] or by secondary structures generated in long gap regions of the lagging strand template.

Replication on the pONE_nick Substrate

A complete lagging strand cycle can be divided into two phases, the replication phase and the interval phase where the primer handoff process is executed. The ability of the replisome to synthesize longer Okazaki fragments when the handoff process is delayed, for example when clamp or clamp loader are diluted [

], indicates that the helicase keeps unwinding during the interval phase. As a result, the size of the lagging strand loop template generated would surpass that of the penultimate Okazaki fragment. Thus, with the collision model, the Okazaki fragment would progressively become longer unless the rate of polymerase replication of the lagging strand is faster than that of helicase unwinding or an RNA primer generated in parallel with ongoing lagging strand synthesis is captured by the lagging strand polymerase before dissociating from the DNA template. The latter possibility is greatly reduced by using a substrate with only one priming site per 1.1 kb [Figure 3A], as opposed to the normal substrates with numerous priming sites. Because we have no evidence that the lagging strand polymerase replicates DNA at a rate greater than that of the leading strand polymerase [see also the Discussion], use of this substrate should reveal the longer Okazaki fragments predicted by the collision model.

Figure 3Replication Reactions on pONE_nick Substrate

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[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.

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The substrate we designed [pONE_nick] contains numerous 5′GCT priming sites but only a single usable 5′GTT site. In the presence of all four rNTPs, many priming sites can be utilized and this substrate behaves like any nicked duplex DNA substrate [Figure 3B]. However, when only ATP and CTP are present, pentamer synthesis by the primase is limited to the single 5′GTT site and a ladder of lagging strand products was observed on the gel [Figure 3C]. The lowest band has a size of 1.1 kb, corresponding to the size of the substrate. The upper bands represent lagging strand products formed either when the primase skipped priming sites or the primer synthesized was not picked up for initiating Okazaki fragment synthesis. This pattern of lagging strand synthesis is expected when the replisome encounters one priming site per 1.1 kb. The distribution of Okazaki fragments of different sizes, however, remains the same over the time course of the reaction [Figure 3D], an observation inconsistent with the collision model.

If collision of the holoenzyme with the 5′ end of the previous Okazaki fragment is the only signaling mechanism for the recycling of the lagging strand polymerase, replication forks should be unable to shift to synthesizing shorter Okazaki fragments when travel of the fork remains unchanged. We have shown previously that longer Okazaki fragments were generated when rNTPs were diluted [

]. When a replication reaction on the pONE_nick substrate was carried out at lower [CTP] [5 μM] in the absence of GTP and UTP, longer Okazaki fragments were synthesized with little 1.1 Kb product [Figure 4A]. Upon addition of 100 μM CTP to the ongoing reaction, the shorter 1.1 Kb fragment was observed within 30 s [Figure 4B]. To ensure that the 1.1 Kb fragment was not synthesized from new active forks formed after the addition of more CTP, we diluted the pONE_nick substrate 10-fold upon addition of CTP. As demonstrated previously [

], the introduction of the dilution step prevents new active replisome formation [data not shown]. This experiment indicates that the replication fork can readily shift from synthesizing long Okazaki fragments to shorter ones, an observation that can be easily explained by the signaling model, but not the collision model.

Figure 4Replication Reactions on pONE_nick Substrate with Varying Amounts of CTP

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[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.

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Primer Utilization Increases at Increasing Concentrations of Clamp and Clamp Loader

The results presented so far argue against the collision model as the only mechanism for release and recycling of the lagging polymerase. All the results, however, can be accommodated by the signaling model. In this model, the true signal can be any of the steps before lagging strand holoenzyme reassembly, such as the priming events [reassociation of the primase with the lagging strand template and subsequent primer synthesis] or the loading of the clamp onto the RNA primer. We have shown previously that the dilution of the clamp and the clamp loader significantly delays the primer handoff process. The continued unwinding by the helicase results in the formation of longer lagging strand template loops and hence longer Okazaki fragments [

]. If the priming events serve as the lagging strand signal, diluting clamp/clamp loader should not decrease the frequency of the signal. Hence, the tempo of lagging polymerase recycling, as well as the size of the Okazaki fragments, should not change. The only change that one expects to observe is longer gaps on the lagging strand due to longer lagging strand template loops. Therefore, the previously observed increase in the size of Okazaki fragments at lower clamp/clamp loader concentrations suggests that the clamp loading is the trigger for lagging polymerase release.

If the loading of clamp onto the newly synthesized RNA primer serves as the signal for lagging polymerase release, then the concentration of the clamp and the clamp loader should affect the efficiency with which the RNA primers are utilized for Okazaki fragment synthesis. To test this, we studied primer utilization at various clamp and clamp loader concentrations. All eight T4 replisomal proteins were tested to be free of RNase contamination. [α-32P]CTP was included in the reactions to label the RNA primers, which were analyzed on a 20% denaturing acrylamide gel. The unused free pentaribonucleotide primers ran into the gel, whereas the primers that had been extended for Okazaki fragment synthesis were retained in sample wells due to their larger sizes. The primer utilization was calculated directly from the proportion of the utilized primers [those incorporated into the Okazaki fragments] and the total primers synthesized [utilized and free primers]. In a control reaction, polymerase was omitted to evaluate the background priming, which was then subtracted from the free primers measured in all reactions.

As shown in Figure 5A, with increasing clamp/clamp loader concentrations, the amounts of free primers decrease, whereas the amounts of utilized primers increase, resulting in an increase in primer utilization from 0.1% to 36.5%. Because we have observed background priming [priming outside the replisome] when using the minicircle substrate, we repeated the same experiments on the pGEM_nick substrate where we showed no background priming in the absence of DNA synthesis [data not shown]. Similar results were obtained when using the larger pGEM_nick substrate [9.7%–40%, Figure 5B]. These results therefore demonstrate the importance of the clamp and the clamp loader in capturing the RNA primers and suggest that the loading of the clamp onto the RNA primer is the actual signal for the recycling of the lagging strand polymerase. Although, the total number of primers synthesized decreases at increasing clamp/clamp loader concentrations, suggesting that priming activity and accessory proteins levels are not independent [Figure S4], the decrease in total priming is not the cause of the higher primer utilization. Varying the concentration of primase [and therefore the total priming activity] has no effect on primer utilization [Figure S5].

Figure 5Effect of Clamp and Clamp Loader Concentrations on Primer Utilization and the Size of Okazaki Fragments

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[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.

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If clamp loading triggers polymerase recycling, then the primer handoff in T4 lagging strand synthesis should follow an indirect process in which the loading of the clamp on the RNA primer precedes the loading of the polymerase. This is different from the direct handoff model proposed in the T7 system in which the primer handoff process was mediated through a direct primase/polymerase interaction [

].

Okazaki Fragment Size Decreases at Increasing Clamp/Clamp Loader Concentrations

A direct consequence of the clamp loading as the trigger for lagging strand polymerase recycling is that high clamp/clamp loader concentrations should induce more frequent lagging strand polymerase recycling and hence the formation of shorter Okazaki fragments. To test this idea, replication reactions were carried out on the pGEM_nick substrate at increasing clamp and clamp loader concentrations. As shown in Figure 5C, the peak of the Okazaki fragment size distribution decreases from a normal size of 1.8–0.84 kb when the amount of clamp and clamp loader increases from 0.06 to 1 μM. This result provides further support for the loading of the clamp onto the primer being the signal for the recycling of the lagging polymerase.

Markov Chain Monte Carlo Simulations of Okazaki Fragment Size

In order to determine whether an experimentally observed distribution of Okazaki fragment sizes can be explained by a signaling mechanism, we chose to simulate the distribution of Okazaki fragments reported for the T4 replisome acting on M13 in vitro as determined by electron microscopy. We modeled a single replisome performing repeated lagging strand synthesis by using the following kinetic scheme: [1] P + D ↔ PD: k1 = 2.0 μM−1s−1, k2 = 0.1 sec−1; [2] PD → DR + P: k3 = 0.75 sec−1; [3] DR → D + R: k4 = 0.2 sec−1; [4] DR + C ↔ DRC: k5 = 0.6 μM−1s−1, k6 = 0.3 sec−1; [5] DRC → DRC∗: k7 = 1 sec−1; and [6] DRC∗ → O + D + C: k8 = 100 sec−1, where P, D, R, C, and O represent primase, lagging strand template, RNA pentamer, clamp/clamp loader, and Okazaki fragment, respectively. We are assuming that these steps occur in parallel with helicase unwinding, so that the total time taken to cycle through the pathway is equal to the amount of time helicase spends unwinding duplex DNA [which governs the length of the lagging strand template]. As a consequence of this assumption, if the helicase paused during any of the above steps, that step would not contribute to the size of the Okazaki fragment. Several of the above rate constants were constrained to values consistent with previous experimental results. The kon and koff for the primase binding to DNA are not known; however, the Kd has been determined to be between 0.05 and 0.1 μM [

], which agrees well with the ratio of k2 over k1. The priming rate of primase [k3] was taken from the same work. The rate of primer capture by the clamp and its dissociation [k5 and k6, respectively] were taken from the rate constants determined for DNA/DNA primer templates [

]. Other rate constants [k4 and k7] were fitted through manual optimization in order to achieve a close correspondence between the simulated and experimental data for Okazaki fragment size distribution [

] and efficiency of primer utilization [this work]. Step 5 [k7] represents the intramolecular rate constant for release of the lagging strand polymerase from the ongoing Okazaki fragment and recycling to the next clamp loaded RNA primer. Step 6 [k8] was included to reflect the distributive nature of the clamp protein and does not contribute to the overall rate of the pathway. One assumption of this scheme is that primase always has a priming site available, which is the case in the M13 substrate.

Figure 6A shows the raw output from the stochastic simulation of the kinetic scheme overlaid with the deterministic solution calculated by numerical integration. The agreement validates the stochastic approach. The delay between the integer increases in Okazaki fragment numbers indicates the time for each Okazaki fragment cycle. It is important to note that the delay time represents the distribution of primers poised for extension [DRC∗]. Assuming an indirect primer handoff, the release of the lagging strand polymerase can occur either by collision [when lagging strand template is replicated prior to primer capture by the clamp] or by signaling [when primer capture by the clamp occurs prior to complete replication of the lagging strand template]. In the former case, k7 represents the time of holoenzyme assembly. In the latter, it is assumed that any ssDNA gaps that may form are filled in to give the final observed Okazaki fragment length distribution. Hence, the collision model falls within the context of the signaling model, as described above. As shown in Figure 6B, stochastic simulation of the signaling mechanism using the above rate constants produces a distribution of Okazaki fragments very similar to the in vitro distribution for the T4 replisome acting on an M13 [

]. The peak of the histogram for the simulated data is 900 bp, which is markedly shifted from the weighted mean average value of 2737 bp, indicating that the predicted Okazaki fragment distribution is clearly not Gaussian in nature and is in accord with the previously noted experimental non-Gaussian distribution of Okazaki fragment sizes [

]. In addition to the simulation corresponding to the experimental data under standard conditions, Okazaki fragment size distribution and primer utilization are affected in the expected manner upon changes in concentration of clamp/clamp loader or primase [Table S1].

Figure 6Okazaki Fragment Size Distribution Determined by Markov Chain Monte Carlo Simulation

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[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

.

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The Standard Replication Reactions

Replication reactions were carried out in a complex buffer containing 25 mM Tris-acetate [pH 7.5], 125 mM KOAc, and 10 mM Mg[OAc]2. The standard replication conditions used in all minicircle reactions as well as the assay methods were essentially as described previously [

]. The same reaction and quenching conditions were also applied to the nicked pONE_nick substrate except that the DNA concentration was at 5 nM and [α-32P]dCTP was added 7 min after reaction. The rolling circle replication reactions using pGEM_nick were carried out in the standard replication buffer containing 5 nM pGEM_nick, 200 nM gp43, 400 nM gp41, 400 nM gp61, 100 nM gp59, 4 μM gp32, 100 μM each of CTP, GTP, and UTP, 2 mM ATP, and 100 μM dNTPs, in a typical reaction volume of 45 μl. The concentrations of gp45 and gp44/62 can be found in the figure legends. Replisome assembly and DNA replication were allowed to proceed for 7 min before the addition of 10 μCi of [α-32P]dCTP. The reaction was quenched with an equal volume of 500 mM EDTA at 10 min and analyzed by 0.8% alkaline agarose gel electrophoresis.

Extension of the Okazaki Fragments by Thermophilic Polymerases

Standard minicircle replication reactions as described in the previous section were carried out at dCTP concentrations of 2.5, 10, and 50 μM. The DNA products were purified by Qiaquick spin columns. The DNA products were extended for 30 min at 72°C by either pfuTurbo [Stratagene] or Deep Vent polymerase [New England Biolabs] in the presence of 250 μM dNTPs. Extension products were analyzed by 0.8% alkaline agarose gel electrophoresis.

Measurement of the Primer Utilization

Standard replication reactions were carried out at various gp45/gp44/62 concentrations [0.025, 0.1, 0.25, 0.5, and 1 μM with the minicircle substrate and 0.0625, 0.125, 0.25, 0.5, and 1 μM with the pGEM_nick substrate]. [α-32P]CTP was included in the reactions for the detection of the RNA primers and was added 1 and 7 min after the initiation of the reaction for minicircle and pGEM substrates, respectively. The reactions were allowed to proceed for another 3 min before being quenched in an equal volume of 0.5 M EDTA. Reaction products were analyzed by electrophoresis on a 20% denaturing acrylamide gel.

Markov Chain Monte Carlo Simulations of Okazaki Fragment Size

The stochastically determined rates of lagging strand polymerase recycling were generated by Dizzy Software [Institute of Systems Biology, Seattle; [

]]. The Gillespie algorithm was used, which simulates chemical reactions as Markovian processes that occur with a certain probability based on kinetic rate constants and the concentration of the components in the system [

]. The protein concentrations used for rate constant optimization were 200 nM clamp/clamp loader, 100 nM DNA template, and 600 nM primase. Processive components of the replisome were stoichometric with the DNA template. To generate histograms of Okazaki fragment size, the output from the simulation was converted to a series of delay times between the production of Okazaki fragments and multiplied by the rate of replisomal DNA synthesis [150 bp/s]. Dynafit software [

] was used to numerically integrate the deterministic differential rate equation.

Publication History

Published: January 19, 2006

Accepted: November 29, 2005

Received in revised form: October 27, 2005

Received: August 2, 2005

Identification

DOI: //doi.org/10.1016/j.molcel.2005.11.029

Copyright

© 2006 Elsevier Inc. Published by Elsevier Inc.

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