(f) Curve-fit analysis is shown for the for Pol III* dissociation in the presence of dNTPs over a 4-nt gapped template: data (open circles), curve fit to single exponential decay (red), curve fit to double exponential decay (cyan).ДЄn exogenous primed template and ATP accelerate Pol III* release from templates with filled gaps. The data shown is only for the first h after injection. Dissociation was allowed to continue for three days. (c) (d) and (e) Injection of either buffer (red) or dNTPs (green) over the 4-nt gapped template 5-nt gapped template, and 10-nt gapped template, respectively. A second injection contained either buffer or a solution containing 40ВµM dCTP, dTTP, and ddGTP (dNTPs) was made at 540 s. (b) Initiation complexes were assembled onto the immobilized DNA primer-template by injecting Pol III*, ОІ 2 and ATP. The 3вЂІ-end of the blocking oligonucleotide was covalently linked to the SNAP protein to prevent ОІ 2 from sliding off the end. (a) Three primed templates containing 4-, 5- and 10-nt gaps were anchored to the surface of a streptavidin-coated sensor chip using biotin-labeled oligonucleotides. The dissociation of Pol III* from primed templates was measured using surface plasmon resonance. Pol III* does not release rapidly upon filling a gap. Only the О± subunit cross-linked, indicating that it serves as the sensor.Д¬opyright В© 2011 Elsevier Ltd. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-cross-linking experiments with highly reactive and non-chemoselective diazirines. Modest (approximately 7 nt) strand displacement is observed after the gap between model Okazaki fragments is filled. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Thus, the rate of polymerase release upon gap completion and collision with a downstream Okazaki fragment is 1000-fold too slow to support an adequate rate of cycling and likely provides a backup mechanism to enable polymerase release when the other cycling signals are absent. Neither a 5'-triphosphate nor a 5'-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. An exogenous primed template and ATP accelerated dissociation an additional 4-fold in a reaction that required complete filling of the gap. Reduction in gap size to 1 nt increased the rate of dissociation 2.5-fold, and complete filling of the gap increased the off-rate an additional 3-fold (t(1/2)~2 min). Initiation complexes exhibit a half-life of dissociation of approximately 15 min. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5'-end of the preceding Okazaki fragment.

Upon completion of synthesis of an Okazaki fragment, the lagging strand replicase must recycle to the next primer at the replication fork in under 0.1 s to sustain the physiological rate of DNA synthesis.

7 strand fill the gap braid