Cooperative base pair melting by helicase and polymerase positioned one nucleotide from each other
- Rutgers-Robert Wood Johnson Medical School, United States
Figures
Figure 1 with 3 supplements
Kinetic mechanism of DNA unwinding by T7 DNAP.
(A) Preformed replication fork DNA substrate for the measurement of the unwinding kinetics of T7 DNA polymerase (DNAP). (B) Representative kinetics of DNA unwinding by T7 DNAP in the presence of E. …
Figure 1—figure supplement 1
Unwinding by T7 DNAP with gp2.5 and SSB.
(A) Unwinding rate of T7 DNAP with T7 gp2.5 (3 μM) and E. coli SSB (200 nM) of the 20% GC DNA substrate at 50 μM dNTPs concentration. Both T7 gp2.5 and E. coli SSB can stimulate the unwinding rate …
Figure 1—figure supplement 2
Unwinding trace of T7 DNAP with SSB.
(A) The kinetic trace for strand displacement DNA synthesis using the fluorescence-based assay shows an initial time lag followed by fluorescence increase in two phases. The traces shown are using …
Figure 1—figure supplement 3
Fitting kinetics of unwinding by T7 DNAP with SSB.
The rate dependencies of the T7 DNAP as a function of dNTPs concentration for dsDNA of increasing GC content were fit to the minimal mechanism shown above to estimate the K1 values shown in Figure 1G…
Figure 2 with 2 supplements
Kinetic mechanism of DNA unwinding by T7 helicase.
(A) Replication fork DNA for the measurement of the unwinding kinetics of T7 helicase. (B) Representative kinetic trace of DNA unwinding by T7 helicase (dots) fit to the n-step model (solid line). (C…
Figure 2—figure supplement 1
Fitting kinetics of unwinding by T7 helicase.
The dTTP dependency data for unwinding by the helicase on the 20–65% GC DNA were fit to the model A (A) model B (B and C) and model C (D). The data do not fit well to model A. Although it fits …
Figure 2—figure supplement 2
Pi release kinetics.
Burst Rate of Pi release plotted as a function of dTTP to obtain the Km, dTTP in the presence of dT90 ssDNA. The error represents fitting error.
Figure 3 with 1 supplement
The kinetics of DNA unwinding by the combined helicase and DNAP enzymes.
(A) The replication fork DNA substrate and representative kinetic trace of DNA unwinding by the combined T7 DNAP and T7 helicase enzymes. (B) The base pair unwinding rates of the combined enzymes at …
Figure 3—figure supplement 1
Unwinding-synthesis trace for helicase-DNAP.
(A) Representative trace showing time course of unwinding-synthesis by the helicase–polymerase on the 20% GC and 50% GC DNA substrate at 100 μM dTTP and 5 μM dVTP. The dip in signal is more …
Figure 4
Functionally coupled and uncoupled helicase-DNAP.
(A) The unwinding rates of the combined helicase-DNAP were measured at 50 µM dTTP (red circle) or 500 µM dTTP (green circle) at increasing dVTPs concentration on the 50% GC fork. The bar chart shows …
Figure 5 with 5 supplements
Base pair melting by isolated and combined T7 DNAP and T7 helicase using 2-aminopurine fluorescence changes.
(A) Structure of the 2-aminopurine (2-AP):T base pair. (B) Structure of the replication fork substrate for 2-AP studies. The primer-end is N and subsequent base pairs are N + 1, N + 2, etc. The …
Figure 5—figure supplement 1
Determining optimal conditions for T7 DNAP and extent of influence of T7 DNAP on template bases in replication fork substrate.
(A) Effect of increasing ratio of the T7 DNAP processivity factor thioredoxin on the 2-AP fluorescence signal. A final ratio of 1:2.5 was used in all the experiments. (B) Effect of DNAP …
Figure 5—figure supplement 2
Effect of T7 DNAP on 2-AP at the junction at N + 2 and N + 3.
(A and B) Fluorescence intensities of fork DNA with 2-AP part of the junction base pair at A. N + 2 and B. N + 3 positions in the lagging strand with and without T7 DNAP. The cartoons show the …
Figure 5—figure supplement 3
Base pair melting by combined SSB—DNAP using 2-AP fluorescence change.
Fluorescence intensities of 2-AP modified replication fork substrates with and without T7 DNAP and E. coli SSB. The cartoons show the structure of fork DNA before and after binding of combined T7 …
Figure 5—figure supplement 4
Determining optimal conditions for T7 helicase.
(A) Effect of helicase concentration on fluorescence emission of 2-AP in the +1 lag 1 gap substrate. Final enzyme concentration of 200 nM was used in all experiments. (B) Fold change on helicase …
Figure 5—figure supplement 5
(A–C) Fluorescence intensities of fork DNA with 2-AP in the lagging strand at the fork junction and increasing distance between primer end and fork junction with (red bars) and without T7 helicase (blue bars).
The cartoons show the structure of fork DNA before and after binding of T7 helicase. The numbers above the bar refer to fold change of 2-AP intensity on protein binding to the DNA. (D) Fluorescence …
Figure 6
Proposed model of DNA unwinding-synthesis by T7 replisome.
The top cartoon of T7 replisome results after melting of the N + 2 base pair by helicase and DNAP. There are two unwound nucleotides (N + 1 and N + 2) between the primer-end and fork junction at N + …
Author response image 1
Additional files
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Supplementary file 1
DNA sequences used in unwinding and 2-aminopurine experiments.
- https://doi.org/10.7554/eLife.06562.020
