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Cycling of the Lagging Strand Replicase During Okazaki Fragment Synthesis

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Cycling of the Lagging Strand Replicase During Okazaki Fragment Synthesis Molecular Life Sciences DOI 10.1007/978-1-4614-6436-5_132-1 # Springer Science+Business Media New York 2014 Cycling of the Lagging Strand Replicase During Okazaki Fragment Synthesis Charles McHenry* Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO, USA Synopsis The E. coli replicase has enormous processivity, sufficient to synthesize over 100,000 bases without dissociation. Yet, on the lagging strand of the replication fork, Okazaki fragments of 1,000–2,000 bases are made every 1–2 s. This requires a mechanism to trigger the lagging strand polymerase to cycle in far less than one second to new primers synthesized at the replication fork. Several mechanisms have been proposed to explain how this might occur. The most prominent have been the collision model where a direct collision with the 50-end of the preceding Okazaki fragment triggers cycling and the signaling model where synthesis of a new primer at the fork triggers polymerase release and rebinding to the new primer. Kinetic studies indicate release after collision is far too slow to support the rate of lagging strand synthesis. Experimental support for the signaling model has been obtained on synthetic mini-circle templates that contain high asymmetry in GC composition between the two strands, allowing the rate of lagging strand synthesis to be selectively perturbed. Furthermore, cycling can be induced by addition of exogenous primers, indicating that it is the presence of a new primer, not the action of primase, that provides the signal. Introduction The Pol III HE has the processivity required to replicate >150 kb (Mok and Marians 1987a, b) and perhaps the entire E. coli chromosome, without dissociation, yet it must be able to efficiently cycle to the next primer synthesized at the replication fork upon the completion of each Okazaki fragment at a rate faster than Okazaki fragment production. The rate of replication fork progression in E. coli is about 600 nt/s at 30 C (Breier et al. 2005), approximately the rate of replicase progression on single- stranded templates (Johanson and McHenry 1982). Thus, most of the time in an Okazaki fragment cycle is spent on elongation and little time (0.1 s) remains for the holoenzyme to release, bind the next primer, and begin synthesis. A processivity switch must be present to increase the off-rate of the lagging strand polymerase by several orders of magnitude. There are two competing but nonexclusive models for the signal that throw the processivity switch. The first (signaling model) proposes that a signal is provided by synthesis of a new primer at the replication fork that induces the lagging strand polymerase to dissociate, even if the Okazaki fragment has not been completed (Wu et al. 1992b). The second (collision model) was originally proposed for T4 (Alberts et al. 1983) and then extended to the E. coli system (Leu et al. 2003). The collision model posits that the lagging strand polymerase replicates to the last nucleotide (Leu et al. 2003) or until the Okazaki fragment is nearly complete (Georgescu et al. 2009). A communication circuit that proceeds through the t subunit has been proposed to sense the conversion of a gap to a nick, signaling release. *Email: [email protected] Page 1 of 9 Molecular Life Sciences DOI 10.1007/978-1-4614-6436-5_132-1 # Springer Science+Business Media New York 2014 A third model for cycling has been proposed that attributed dissociation of the lagging strand polymerase induced by the inability of the dimeric polymerase to rotate around the template strand once per helical turn, resulting in highly supercoiled product (Kurth et al. 2013). Shortly after the discovery that t dimerizes the leading and lagging strand polymerases, it was recognized that negative superhelical torque could be an issue for the leading strand, but that rotation within single-stranded DNA would rapidly dissipate superhelical tension in the lagging strand (McHenry et al. 1988). However, Marians observed that coupled leading and lagging strand rolling circle replication could generate leading strand products of approximately 150,000 bases without the addition of topoisomerases (Mok and Marians 1987a, b). This suggested that if this torque was generated, it could be relieved by mechanisms that did not involve topoisomerase action. Cozzarelli and colleagues considered the issue in more detail (Ullsperger et al. 1995) and discounted the notion of extensive precatenane interwinding of the leading and lagging strand product created by rotation of the lagging strand polymerase about the leading strand to remove the torque. As one plausible solution, they proposed the 30-end of the growing leading strand could occasionally be released from the active site of the polymerase to allow dissipation of superhelicity (Ullsperger et al. 1995). It would appear that the ring-like structure of the b sliding clamp might allow rotation of the helix through its central pore without dissociation, maintaining processive leading strand synthesis. Another possible mechanism for torque release would be dissipation of leading strand torque by release of the lagging strand polymerase during cycling to the primer for synthesis of the next Okazaki fragment. This mechanism would appear cumbersome, but not impossible, because of the large mass and radius of gyration of the Pol III HE in the viscous milieu of the cell. However, if cycling involves DnaX as the sensor that initiates cycling (section Cycling of the Lagging Strand Polymerase in E. coli is Directed Exclusively by a Modified Signaling Model), it would be attached to the priming apparatus, precluding this mechanism. Evidence that a modification of the signaling model is exclusively used to drive cycling in E. coli is presented in the last section of this essay. Cycling Models Provided by Bacteriophages T4 and T7 In the more fully characterized systems provided by the replication apparatus of bacteriophages T4 and T7, signaling through synthesis or the availability of a new primer appears to play an important role, with the collision pathway playing a backup role (Hamdan et al. 2009). A handoff of the nascent tetraribonucleotide primer and the T7 polymerase is effected by direct primase-polymerase interaction (Kato et al. 2004). It takes time for synthesis of a new primer, release of the lagging strand polymerase, and initiation of synthesis on a new primer. Differing views have been presented on how T7 overcomes this delay on the lagging strand to allow leading and lagging strand replication to remain coordinated (Lee et al. 2006; Pandey et al. 2009). In one model, it is proposed that the helicase and thus leading strand synthesis is halted during slow primer synthesis (Lee et al. 2006). In the other model, it is proposed that the lagging strand primer is synthesized before it is needed and held in a priming loop, close to the replication fork, facilitating handoff to the DNA polymerase (Pandey et al. 2009). This model, if generally applicable, could explain the double loops sometimes observed at replication forks by electron microscopy (Chastain et al. 2003). In the latter model, it is proposed that the reactions remain coordinated because the lagging strand polymerase elongates faster than the leading strand polymerase (Pandey et al. 2009). In T4, primer synthesis does not halt progression of the leading strand polymerase. Primer synthesis occurs by two mechanisms: (1) dissociative, with primase releasing from its helicase Page 2 of 9 Molecular Life Sciences DOI 10.1007/978-1-4614-6436-5_132-1 # Springer Science+Business Media New York 2014 association during primer synthesis, or (2) processive, whereby primase remains associated and a second loop is formed on the lagging strand DNA (Manosas et al. 2009; Yang et al. 2006). The sliding clamp and clamp loader increase the processive/looping mechanism and it was suggested that gp32 (T4 SSB) might further increase the processive looping mechanism in the natural system by facilitating handoff of the nascent pentaribonucleotide primer (Manosas et al. 2009). A proposal was made that the clamp loader/clamp interaction with a new primer might be the key signal required for release of the lagging strand polymerase (Yang et al. 2004). More recent work supports that proposal (Chen et al. 2013). The size distribution of short Okazaki fragments that derive from signaling-induced cycling are affected by concentrations of the clamp loader while independent of polymerase concentration, as expected for a processive, recycled lagging strand polymerase. Does the OB Fold Provide the Processivity Switch for Cycling? In the structure of Pol III a complexed to DNA, an OB fold is located close to the primer terminus (Wing et al. 2008). Because OB folds commonly bind to ssDNA, a proposal was made that it could be part of the sensing network (Bailey et al. 2006; Wing et al. 2008). Consistent with this hypothesis, the ssDNA binding portion of Pol III was localized to a C-terminal region of a that contains the OB fold element (McCauley et al. 2008). A test of the importance of the OB fold motif was made using a mutant in which three basic residues located in the b1-b2 loop were changed to serine (Georgescu et al. 2009). No ssDNA binding was observed in the mutant, indicating diminution in affinity. However, even the wild-type polymerase bound ssDNA extremely weakly, near the limit of detection in the assays used (Kd 8 mM). The processivity of the mutant polymerase was decreased by the b1-b2 loop mutations, an effect that was rescued by the presence of the t-complex (Georgescu et al. 2009). The latter observation would seem to suggest that although the OB fold contributes to ssDNA affinity and processivity, it is not the processivity sensor or at least that the residues mutated are not the key interactors.
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