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Review
. 2016 Oct 1;108:65-78.
doi: 10.1016/j.ymeth.2016.05.003. Epub 2016 May 9.

Methods to Study the Coupling Between Replicative Helicase and Leading-Strand DNA Polymerase at the Replication Fork

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Free PMC article
Review

Methods to Study the Coupling Between Replicative Helicase and Leading-Strand DNA Polymerase at the Replication Fork

Divya Nandakumar et al. Methods. .
Free PMC article

Abstract

Replicative helicases work closely with the replicative DNA polymerases to ensure that the genomic DNA is copied in a timely and error free manner. In the replisomes of prokaryotes, mitochondria, and eukaryotes, the helicase and DNA polymerase enzymes are functionally and physically coupled at the leading strand replication fork and rely on each other for optimal DNA strand separation and synthesis activities. In this review, we describe pre-steady state kinetic methods to quantify the base pair unwinding-synthesis rate constant, a fundamental parameter to understand how the helicase and polymerase help each other during leading strand replication. We describe a robust method to measure the chemical step size of the helicase-polymerase complex that determines how the two motors are energetically coupled while tracking along the DNA. The 2-aminopurine fluorescence-based method provide structural information on the leading strand helicase-polymerase complex, such as the distance between the two enzymes, their relative positions at the replication fork, and their roles in fork junction melting. The combined information garnered from these methods informs on the mutual dependencies between the helicase and DNA polymerase enzymes, their stepping mechanism, and their individual functions at the replication fork during leading strand replication.

Keywords: Bacteriophage T7 replication; Base pair unwinding-synthesis rate constant; Chemical step size; DNA polymerase; Mitochondrial DNA replication; Pre-steady state kinetics; Replicative helicase; Structural biology.

Figures

Figure 1
Figure 1. Helicase - DNA polymerase at the replication fork
(A) Cartoon showing the hexameric ring shaped replicative helicase and the replicative DNA polymerase enzymes bound to a replication fork DNA that represents an intermediate structure during leading strand replication. (B & C) Two proposed models for the architecture of the eukaryotic replisome based on single particle EM studies [15]. Depending on the orientation of the helicase with respect to the fork junction (If the C-terminal domain of the helicase is facing the fork junction the leading strand DNAP is present ahead as in (B) or if the N-terminal domain of the helicase is facing the fork junction the leading strand DNAP is behind the helicase as in (C). Further studies that capture the DNA path are required to identify the correct orientation of the system.
Figure 2
Figure 2. Replication Fork Substrate
Cartoon of the replication fork DNA mimic made from chemically synthesized oligodeoxynucleotides. The lagging strand DNA is shown in blue, the leading strand DNA in red and the primer strand in green.
Figure 3
Figure 3. Gel-based assay to determine the base pair unwinding-synthesis rate constant of the helicase-DNAP complex
(A) The helicase-DNAP complex is assembled on the replication fork DNA. The 5’-end of the 24-mer primer in the fork DNA is labeled with a fluorophore or γ[32Pi]. The unwinding-synthesis reaction initiated with dNTPs is monitored by following the extension of the labeled DNA primer. (B) Schematic of the rapid quench-flow instrument for rapid mixing and quenching of the reactions with millsecond time resolution. The duration of the reaction is changed by adjusting the volume of the delay line and the flow rate through the delay loop. (C) Image of polyacrylamide/urea sequencing gel showing extension of the 24-mer DNA primer by T7 helicase-DNAP (1 mM dNTPs) at 18 °C. The T7 helicase is the product of T7 gp4, which contains both helicase and primase activities[58]. T7 DNAP is a 1:1 complex of polymerase T7 gp5 and processivity factor E. coli thioredoxin[59]. (D) The polymerization model is used to globally fit the formation and decay of each primer extension product to obtain the individual base pair unwinding-synthesis rate constants. The kp1, kp2, etc. are the rate constants for the formation of Dn+1, Dn+2, etc. extension products, respectively. The ko1, ko2, etc. are the rate constants for the dissociation of the DNAP from the Dn+1, Dn+2, etc. complex intermediates. The computer fitting provides the rate constants for the individual nucleotide addition reactions (kp1, kp2, etc.) from which one can calculate the average single base pair unwinding synthesis rate constant. (E) The average unwinding-synthesis rate constant of the helicase-DNAP is compared to the helicase’s rate constant of unwinding on fork DNA substrates with 5–80% GC-content. (F) The sequencing gel image shows the strand-displacement DNA synthesis activity of the isolated T7 DNAP (1 mM dNTPs) at 18 °C.
Figure 4
Figure 4. Stopped-flow fluorescence assay to determine the base pair unwinding synthesis kinetics
(A) The replication fork DNA is labeled with the fluorescein fluorophore at the 3’ end of the lagging strand and BHQ-1 quencher at the 5’ end of the leading strand. The fluorescence intensity is quenched when the replication fork DNA is duplexed and increases when the strands are separated by the unwinding-synthesis activity of the helicase-DNAP (B) Schematic of the stopped-flow instrument (KinTek Instrument) that allows rapid mixing of small volumes of reactants and real-time fluorescence intensity measurements with close to 1 ms dead time under temperature controlled conditions. (C) Kinetic traces of 50% GC fork DNA strand separation reaction at 18 °C by the unwinding-synthesis activities of the helicase-DNAP at increasing concentrations of dVTP (dATP + dCTP + dGTP) and 500 µM dTTP (for helicase assembly). The lag represents the time taken to unwind-copy the dsDNA region of the fork DNA, and this lag time decreases as the dNTP concentration (DNAP substrate) increases. The dip in fluorescence signal following the initial increase is potentially due to interaction of the fluorophore with the proteins as they reach the end of the DNA strand [23]. The ‘x’ is time to reach half-maximal fluorescence increase. (D) The unwinding-synthesis rate constants can be estimated by fitting the kinetic data in panel C to the stepping model (green bars) or by multiplying the inverse of the lag time (x) and the dsDNA length (blue bars). (E) Unwinding rate constants of T7 DNAP with SSB (red circles) or with helicase (green circles) with increasing concentrations of dNTPs. (E) The unwinding rate constants of T7 DNAP with SSB (blue bars) or with helicase (red bars) as a function of increasing GC content (5–65%) in the dsDNA region.
Figure 5
Figure 5. One-pot assay to measure the chemical step size (bp/NTP hydrolyzed) of the helicase-DNAP catalyzing leading strand DNA synthesis
(A) One-pot assay to measure the base pairs unwound-copied by the T7 helicase-DNAP and dNTP hydrolyzed by the helicase using [α32P]dNTPs, which quantify the kinetics of dNTP hydrolysis to dNDP and the incorporation of dNMP into the DNA. (B) Thin-layer chromatography (PEI-cellulose) shows the separation of [α32P]dNTPs from the corresponding [α32P]dNDPs and DNA (labeled with [α32P]dNMPs). (C) Kinetics of total dNDPs production and total dNMPs incorporation into the DNA by the helicase-DNAP on the 100% AT-rich fork DNA substrate. (D) Kinetics of total dNDPs production and total dNMPs incorporated on the 50% GC-rich DNA substrate.
Figure 6
Figure 6. Exonuclease footprinting to determine the position of T7 helicase on the replication fork substrate in complex with T7 DNAP
(A) The 5’ end of the lagging DNA strand in the fork DNA (with a 36-nt ssDNA tail) is labeled to visualize the protected DNA fragments after digestion with a combination of Exo III and Exo T enzymes. The simulated gel cartoon shows the expected length of the protected DNA fragments when the helicase is bound at the fork junction. The length of the protected DNA fragments (‘X’) is determined by analysis on a high resolution sequencing gel. If X is less than 36-nt, then the helicase is positioned away from the fork junction, if X is 36-nt then the downstream boundary of the helicase is at the fork junction, and if X is greater than 36-nt then the helicase is positioned downstream of the fork junction. (B) Sequencing gel shows the products of ExoIII + ExoT digestion (X = 38-nt) in the presence and absence of proteins (T7 helicase and T7 helicase-DNAP). The length of the protected DNA fragments with the helicase and helicase-DNAP are similar indicating that the position of the helicase on the fork DNA does not change in the presence of the DNAP. The prominent 38-nt central band indicates that the helicase is within 2-nt downstream of the fork junction [21].
Figure 7
Figure 7. Mapping the position of the DNAP active site on the replication fork with the helicase
(A) DNA substrate with an interstrand transplatin DNA crosslink positioned approximately in the middle of the dsDNA region of the fork substrate. Extension of the labeled primer by the helicase-DNAP complex is carried out in the presence of all dNTPs to map the position of the DNAP active site with respect to the transplatin crosslink. (B) DNA substrate with 20-nt ssDNA gap between the primer end and fork junction. T7 helicase is stalled at the fork junction using dTMP-PCP, a non-hydrolysable dTTP analog, and the extension of the primer by the DNAP to the point of the stalled helicase is followed using a labeled primer. (C) High resolution sequencing gel shows the time course of primer extension. T7 DNAP is able to reach all the way to the crosslink point. (D) High resolution sequencing gel shows the time course of the extension of the primer up to the fork junction without stalling at any upstream positions.
Figure 8
Figure 8. 2-AP fluorescence assay to determine the relative contribution of the helicase and DNAP to dsDNA unwinding
(A) Schematics of the 2-AP labeled fork DNA substrates. A single 2-AP probe (indicated in red) is placed at the fork junction or one base pair downstream of the fork junction. The primer end is adjusted to generate fork DNA substrates with no gap, 1 gap, 2 gap, or 3 nt ssDNA gap between the primer end and fork junction. (B) Change in 2-AP fluorescence intensity on protein binding is monitored over a range of wavelengths. The change in 2-AP intensity on protein binding reflects on the effect of the protein on the 2-AP base pair. (C) Fluorescence intensities of 2-AP modified replication fork DNA with (green bars) and without (blue bars) T7 DNAP. Cartoons of the DNA substrate used in the experiment and the fold change of fluorescence intensity on protein binding are shown above each bar. (D) Fluorescence intensities of 2-AP modified replication fork DNA with (red bars) and without (blue bars) T7 helicase. (E) Fluorescence intensities of 2-AP modified replication fork DNA with (light blue bars) and without (blue bars) T7 helicase-DNAP. The red bars show intensity changes with T7 helicase alone. (F) Fluorescence intensities of 2-AP modified replication fork DNA with (light blue bars) and without (blue bars) T7 helicase-DNAP. The red bars are the effect of just T7 helicase binding in the absence of T7 DNAP.

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