The Rate of Polymerase Release upon Filling the Gap between Okazaki Fragments Is Inadequate to Support Cycling during Lagging Strand Synthesis

Abstract

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 second to sustain the physiological rate of DNA synthesis. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5′-end of the preceding Okazaki fragment. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. Initiation complexes exhibit a half-life of dissociation of approximately 15 minutes. Reduction of gap size to one nucleotide increased the rate of dissociation 2.5-fold and complete filling of the gap increased the off rate an additional three-fold (t½ ∼ 2 min). An exogenous primed template and ATP accelerated dissociation an additional four-fold in a reaction that required complete filling of the gap. Neither a 5′-triphosphate nor 5′-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. 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. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Modest (approximately 7 nucleotide) strand displacement is observed after the gap between model Okazaki fragments is filled. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-crosslinking experiments with highly reactive and non-chemoselective diazirines. Only the α subunit cross-linked, indicating it serves as the sensor.

Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes

“…However, if a new polymerase is used for the synthesis of almost every Okazaki fragment, as suggested by previous exchange observations (Geertsema et al, 2014), these pathways are not required. Supporting this line of reasoning, several studies have suggested that no specific protein factor exists for signaling (Kurth et al, 2013), and that collision events are orders of magnitude too slow to support efficient replication (Dohrmann et al, 2011). These findings suggest polymerase release is a stochastic event with many possible triggers.…”

“…Processive replication events reveal polymerases remaining on the lagging strand behind the replisome ( Figure 5B). Some polymerases remain bound on the lagging strand for very long times (minutes) consistent with stalling upon completion of Okazaki-fragment synthesis (Dohrmann et al, 2011;Huber et al, 1987). Examination of kymographs from 55 replication events reveals that polymerase emergence from the replication fork is four times more frequent than direct binding from solution to the lagging-strand ( Figure 5C & S10), and the mean spacing between polymerases is 3.8 ± 0.4 kb, consistent with most polymerases rapidly completing…”

Replicative DNA Polymerases

“…In addition they have either a built-in 3 0 -5 0 exonuclease site, located at a distance from the polymerase active site, or an associated subunit with 3 0 -5 0 exonuclease activity (reviewed in Patel and Loeb 2001;Kunkel 2004;McHenry 2011). In either case, the distance from the polymerase site to the exonuclease site necessitates that at least three nucleotides of the double-stranded DNA are unwound to allow editing.…”

“…As isolated directly from cells, it has an average composition close to (a1u) 2 -(t 2 gdd 0 cx)-(b 2 ) 2 (17 subunits), where a1u is the polymerase core discussed in more detail below, b 2 is the sliding clamp, and t 2 gdd 0 cx is the clamp loader complex that may contain two to three t and one to zero g subunits (McHenry 2011).…”

“…Its primary function in replication is in Okazaki fragment processing on the lagging strand. Pol III is capable of synthesizing Okazaki fragments right up to the 5 0 end of a preceding RNA primer, whereupon it is recycled to a new primer terminus, leaving behind a nick or short gap (discussed in Dohrmann et al 2011). Pol I has three separate activities in a single polypeptide chain.…”