Independent and Stochastic Action of DNA Polymerases in the Replisome

Abstract

SUMMARY It has been assumed that DNA synthesis by the leading- and lagging-strand polymerases in the replisome must be coordinated to avoid the formation of significant gaps in the nascent strands. Using real-time single-molecule analysis, we establish that leading- and lagging-strand DNA polymerases function independently within a single replisome. Although average rates of DNA synthesis on leading and lagging strands are similar, individual trajectories of both DNA polymerases display stochastically switchable rates of synthesis interspersed with distinct pauses. DNA unwinding by the replicative helicase may continue during such pauses, but a self-governing mechanism, where helicase speed is reduced by ~80%, permits recoupling of polymerase to helicase. These features imply a more dynamic, kinetically discontinuous replication process wherein contacts within the replisome are continually broken and reformed. We conclude that the stochastic behavior of replisome components ensures complete DNA duplication without requiring coordination of leading- and lagging-strand synthesis.

What is all this fuss about Tus? Comparison of recent findings from biophysical and biochemical experiments

“…The first two studies demonstrate facile exchange between complete replicase units at the fork and in solution, on the seconds timescale, both in vivo (Beattie et al 2017) and in vitro (Lewis et al 2017). The third study (Graham et al 2017) suggests that even under conditions with no free polymerases in solution, the DnaB helicase can transiently uncouple and proceed more slowly ahead of the replicase at the apex of the replication fork. Although further work is necessary to fully integrate these new findings with our study of the fork rate dependence of replisome stalling at Tus-Ter sites , they do not affect our conclusions.…”

Understanding DNA replication by the bacteriophage T4 replisome

“…The leading-strand polymerase complexed to ␤-clamp and in the presence of DnaB in single-molecule experiments shows bursts of synthesis at 500 -600 nt/s and mean rates of about 350 nt/s independent of DnaG activity (92,93). Again, the activity of DnaB is stimulated by the presence of the leading-strand HE; by itself, the DnaB unwinds duplex DNA at a rate of only 84 -86 bp/s (93).…”

“…Again, the activity of DnaB is stimulated by the presence of the leading-strand HE; by itself, the DnaB unwinds duplex DNA at a rate of only 84 -86 bp/s (93). Consequently, a displacement of about 350 -700 bp between the two polymerases can be imagined as a consequence of each Okazaki fragment synthesized (90).…”

“…An additional hypothesis is that the torsional strain generated by a replisome with two interconnected polymerases is responsible for polymerase recycling (96), but a direct measure of the force required to break the connection would bolster this proposal. Consequently, the literature appears to favor a more conventional signaling mechanism (93,97).…”

“…Recent single-molecule experiments found the replication velocity of the leading-and lagging-strand polymerases to be equivalent, but punctuated with stops/starts to change replication rates (93). Consequently, their behavior would not be coordinated but stochastic.…”