Mechanism of strand displacement synthesis by DNA replicative polymerases

Abstract

Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.

Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity

“…We observe exo-to-pol direct switching, consistent with biochemical and single-molecule data for T7 and T4 DNAp (14,15). In a physiological setting, it would be especially important to escape processive exo and continue replication, since an error is quickly removed and further exonucleolytic activity would be deleterious.…”

“…In accordance with previous studies, we observe that the rate of polymerization decreases with DNA tension up to 35 pN. The polymerization rates we find (up to 500 nt s À1 at a tension of 15 pN) are significantly higher than those previously obtained in bulk (300 nt s À1 ) (6) and optical-tweezers (100 nt s À1 ) experiments (8), but are close to those reported in a recent magnetic-tweezers study (14). The difference between this study and previous optical-tweezers work is likely a consequence of our improved temporal resolution, which allows the identification (and exclusion) of short, previously undetected pauses in-between replication events.…”

“…Surprisingly, we now also observe pol activity at high tension, including tensions close to the dsDNA overstretching transition at~65 pN (19). The polymerization rate in these particular events is relatively low (~80 nt s À1 ) and does not appear to depend on a further increase in tension, in agreement with Manosas et al (14). Our data on T7 DNAp exonucleolysis suggest that it removes correct bases (see also Supporting Materials and Methods) at a constant rate of~150 nt s À1 (n ¼ 4382) over the full range of tensions studied.…”

“…In the case of T7 DNAp, we have found no evidence for such a polymerization state. The model for the family B T4 DNAp that was proposed to be applicable to T7 DNAp entails a kinetic scheme that resembles the scheme presented here (14). This model contains a paused state during polymerization and a direct transition from exo to pol.…”

“…In a single-molecule Förster resonance energy transfer study using fluorescent base analogs, Klenow polymerase was shown to bind relaxed, matched PTS with both its pol and exo active sites (12,13). Moreover, the replicative T4 and T7 DNAp (families B and A, respectively) were previously shown to exhibit similar behavior in preferentially performing exonucleolysis when polymerization was obstructed by dsDNA ahead of the DNAp (14). In addition, processive exonucleolysis was favored over polymerization, only at tensions below 9 pN.…”