Single molecule measurement of the “speed limit” of DNA polymerase

Abstract

Although DNA replication is often imagined as a regular and continuous process, the DNA polymerase enzyme is a complicated machine and can pause upon encountering physical and chemical barriers. We used single molecule measurements to make a detailed characterization of this behavior as a function of the template's secondary structure and the sequence context. Strand displacement replication through a DNA hairpin by single DNA polymerase molecules was measured in real time with near single base resolution and physiological concentrations of nucleotides. These data enabled the measurement of the intrinsic “speed limit” of DNA polymerase by separating the burst synthesis rate from pausing events. The strand displacement burst synthesis rate for Escherichia coli DNA Polymerase I (KF) was found to be an order of magnitude faster than the reported bulk strand displacement rate, a discrepancy that can be accounted for by to sequence specific pausing. The ability to follow trajectories of single molecules revealed that the burst synthesis rate is also highly stochastic and varies up to 50-fold from molecule to molecule. Surprisingly, our results allow a unified explanation of strand displacement and single strand primer extension synthesis rates.

Active DNA unwinding dynamics during processive DNA replication

“…Mechanical extension of the template could prevent base stacking, avoiding in this way the entrance to the short-lived pause state. In fact, other factors known to affect the stability of DNA, as betaine and increasing temperatures, also decrease the pause frequency at GC rich sequences during primer extension conditions for Phi29 and other DNA polymerases, supporting this hypothesis (19,23,24).…”

Modeling translocation dynamics of strand displacement DNA synthesis by DNA polymerase I

“…Even for a very large value of E'=20k B T (see Supporting information), which is equivalent to an equilibrium dissociation constant K d % 2 nM, the mean moving time T m is only 5.96 ms and 1.46 ms for the strand displacement synthesis and single stranded primer extension synthesis, respectively. These values of T m are much smaller than the mean dwell time, T d %71.43 ms, between two successive nucleotide incorporations, which is obtained from the incorporation rate of about 14 s À1 for polymerase I at saturating concentration of dNTP [35]. This implies that the incorporation rate is mainly determined by the chemical reaction rate of the nucleotide incorporation or phosphoryl transfer rather than the moving time.…”

“…The DNA polymerases can also pause due to specific DNA sequences such as palindromic DNA capable of forming hairpin secondary structure [1,23,24], slow zones [2], trinucleotide repeats of (CGG) n (CCG) n or (CTG) n (CAG) n [16,32] and novel sites such as Pyr-G-C [29]. Recently, by using single-molecule techniques, the non-uniform polymerase activity and sequence-dependent pausing during the strand displacement DNA synthesis were demonstrated directly and their dynamics was studied quantitatively [35]. The pauses at the specific sequences have been proposed to be caused by difficulties in the polymerase fingers-closing conformational change, since this transition was thought to be rate-limiting and the most sensitive to changes in temperature [29].…”

“…However, a later experiment [15] showed that the slow prechemistry step is not the fingers-closing transition. Thus, another proposal was that the pauses are associated with an earlier DNA template rearrangement step that might be sequence dependent [35].…”

Topological Interactions in Ring Polymers

“…(C.5) ensures that the un-linked minicircles duplicate at rate α until the sum ρ u + ρ l reaches 2, at which point the growth is stopped, while the third terms describe the usual diffusion of mini-circles and topo II with their respective diffusion constants. The value of α at which mini-circles are duplicated can be estimated by noting that polymerase replicates DNA at a speed between 20 bp/s (in bacteria) and 500 bp/s (in eukaryotes) [Dignam et al, 1983,Wickiser et al, 2005,Schwartz and Quake, 2009. Therefore the typical duplication time results to be of about α −1 2000 bp/(100 bp/s) = 20 s.…”