Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Summary

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Abstract

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Introduction

The replication of DNA is a conserved feature of all cellular life. While the identity and number of replication components vary widely, the general mechanisms are shared across evolution¹. Here, we describe gel-based, discontinuous kinetic experiments, using radioactive substrates, aimed at understanding the kinetics of lagging-strand DNA synthesis in vitro by components of the T7 replisome. The replication machinery of bacteriophage T7 is very simple, consisting of only four proteins (the DNA polymerase, gp5, and its processivity factor, E. coli thioredoxin, the bifunctional primase-helicase gp4, and the single-stranded DNA binding protein, gp2.5)². This feature makes it an attractive model system to study the conserved biochemical mechanisms involved in DNA replication.

Special emphasis is placed on the formation of ribonucleotide primers by T7 DNA primase, a critical step in the initiation of DNA synthesis. In addition, one can also examine the usage of these primers by T7 DNA polymerase or other replication proteins by including them in the reaction mixture. The T7 primase-helicase, gp4, catalyzes the formation of primers for DNA synthesis at specific DNA sequences called primase recognition sites, or PRS, (5'-GTC-3'). The cytosine in the PRS is cryptic, it is essential for recognition of the site, but it is not copied into the product³. The first step in primer synthesis by gp4 involves the formation of the dinucleotide pppAC, which is then extended to a trimer, and eventually to functional tetranucleotide primers pppACCC, pppACCA, or pppACAC depending on the sequence in the template⁴. These primers can then be used by T7 DNA polymerase to initiate DNA synthesis, which is also a gp4-assisted process^5,6. In this regard, the primase domain stabilizes the extremely short tetraribonucleotides with the template, preventing their dissociation, engages DNA polymerase in a manner conducive to securing the primer/template into the polymerase active site⁷. These steps (RNA primer synthesis, primer handoff to polymerase, and extension) are repeated in multiple cycles to replicate the lagging strand and must be coordinated with the replication of the leading strand.

The assays described here are highly sensitive and can be performed in a moderate time frame. However, they are relatively low-throughput and great care must be exercised in the usage and disposal of radioactive materials. Depending on the speed at which the reaction proceeds, one might employ a rapid-quench instrument to attain samples at time scales amenable for meaningful analysis of reaction time courses in either the steady or the pre-steady state. Recently, we used assays described here to provide evidence of the importance of primer release from the primase domain of gp4 in the initiation of Okazaki fragments. Additionally, we found evidence of a regulatory role for the T7 single-stranded DNA binding protein, gp2.5, in promoting efficient primer formation and utilization⁸.

Protocol

NOTE: Follow all institutional regulations regarding the safe use and disposal of radioactive material, including but not limited to, usage of personal protective equipment, such as gloves, safety glasses, lab coat, and appropriate acrylic shields.

NOTE: The standard buffer consists of 40 mM HEPES-KOH, pH 7.5, 50 mM potassium glutamate, 5 mM DTT, 0.1 mM EDTA (this buffer is pre-made at a 5x concentration) and 0.3 mM of each dNTP. T7 replication proteins are purified as described^8,9,10. Single-stranded DNA containing a single T7 primase recognition site (5'-GGGTC-3', 5'-GTGTC-3', 5'-TGGTC-3') can be purchased from a variety of DNA synthesis companies (the length of the ssDNA may depend on the enzyme of choice, for T7 primase, a pentamer is the minimum template length to synthesize a full-length primer¹¹, as the cryptic C is essential, however, if the primer is to be extended by polymerase, additional nucleotides must be present at the 3' end of the template. Reactions are initiated by the addition of a final concentration of 10 mM MgCl₂ and incubated at 25 °C. Manual sampling works well for time-points of 5 seconds or greater. If time-points shorter than that are required, it is recommended to use a rapid-quench flow instrument to obtain accurate data.

1. Multiple-turnover Primer Synthesis Reaction by Manual Sampling

1. Before starting the experiment, aliquot 2 µL of formamide dye buffer (93% (v/v) formamide, 50 mM EDTA, 0.01% xylene cyanol and 0.01% bromophenol blue) into 8 1.5 mL polypropylene tubes. In general, the number of tubes is equal to the number of time-points analyzed.
2. Prepare a 20 µL mixture consisting 1x buffer, 0.1 mM ATP and CTP, 0.25 mCi/mL [α-³²P] CTP, 0.1 µM ssDNA template, and 0.1 µM gp4 (expressed as hexamer concentration — 0.6 µM monomer).
3. Incubate the mixture at room temperature for 5 min.
4. Remove 2 µL of the mixture using a pipettor and mix with 2 µL formamide dye buffer in a 1.5 mL tube prepared in step 1.1 for the no reaction control (time zero).
5. Add 2 µL of 0.1 M MgCl₂ to reaction mixture (to a final concentration of 10 mM).
6. Manually withdraw 2 µL samples of the reaction mixtures using a pipettor at 10 s intervals and mix with formamide dye predispensed in 1.5 mL tubes prepared in step 1.1.
7. Heat samples at 95 °C for 5 min in a heat block.
8. Load aliquots of the samples onto a denaturing gel. The small tetraribonucleotide primer products synthesized by the T7 primase are resolved well on a 0.4 mm thick 25% acrylamide:bisacrylamide (19:1), 3 M urea, 1x TBE (100 mM Tris-borate, 1 mM EDTA).
9. Place entire gel on chromatography paper and cover with plastic wrap. Dry gel using a gel drier.
10. Prepare a dilution series of the remainder of the reaction mixture and spot the same volume loaded on the gel onto chromatography paper, i.e., spot 4 µL of the dilution if 4 µL of sample were loaded.
    NOTE: This will serve as the standard curve to determine the concentration of products formed during the reaction. For example, five-fold dilutions using TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) work well, spanning a range of 1:25 to 1:15,625 (5²-5⁶), but the dilutions should be adjusted depending on the level of activity.
11. Expose dry gel and standard using a phosphorimager screen. Quantify the radioactivity in the dilution series and construct a standard curve as described ^8,12
    NOTE: Inclusion of T7 DNA polymerase to the reaction mixture allows one to examine the usage of tetraribonucleotide primers by this enzyme. Optimally, the polymerase concentration should be saturating to simplify the interpretation of the data. A polymerase concentration of 0.4 µM or higher gives good results. Additionally, the effect of other proteins, such as single-stranded DNA binding proteins, on either primer synthesis or lagging-strand initiation can be examined in this manner.

2. Multiple-turnover Primer Synthesis Reaction Using a Rapid-quench Instrument

1. Preparation of reaction mixtures
   1. Prepare 400 µL of solution A, containing 1x buffer, 0.2 µM gp4 hexamer, 0.2 µM ssDNA template, 0.6 mM dNTPs, 0.2 mM ATP and CTP (including 0.5 mCi/mL [α-³²P] CTP.
   2. Prepare 400 µL of solution B, containing 1x buffer and 20 mM MgCl₂.
2. Operation of rapid-quench flow instrument
   1. Turn on rapid-quench flow instrument; after the main menu appears, type 7 on the instrument keyboard to move the syringe driver to the home position.
   2. Open buffer syringe position to "LOAD".
3. Loading buffer syringes
   1. Attach a 10 mL syringe containing buffer to the drive syringe port. Change syringe knob position to "LOAD".
   2. Using 10 mL syringes, fill instrument buffer reservoir A and B with 10 mM Tris, pH 7.5, 1 mM EDTA and fill syringe C with quench solution (250 mM EDTA, and 0.2% SDS). Remove air bubbles by pushing plungers in and out.
   3. Change knob positions to "FIRE". Type 2 to adjust position of the syringe driver bar. Press "ADJUST DOWN" button to lower the syringe until buffer comes out the exit loop: (7-goes down continuously; 8-large step down; 9-small step down). Type "ESC" to return to the main menu.
4. Wash tubing
   1. Attach exit line to vacuum. Change sample knobs to "FLUSH". Close buffer syringe by setting the knobs to the horizontal position. Turn on vacuum and dip the 2 flush loops in H₂O for 10 s, then dip in MeOH for 10 s. Dry loops by sucking air for ~15 s, or until dry.
5. Loading samples
   1. Change sample knob position to "LOAD". Place solution A into 1 mL syringe. Plug syringe into one of the sample load ports. Repeat for solution B, and place in opposite sample load port.
6. Collection of time-points
   1. On main menu, type 1 for quench-flow run. Enter reaction time (s) and press, "ENTER". The reaction loop number to use will be displayed. Switch to required reaction loop. Repeat wash procedure in step 2.2.4.
   2. Turn sample knobs to "LOAD". Load reaction mixes in sample loops until just outside the central mixing compartment. Turn all knobs to "FIRE". Confirm that all knobs are in the correct position.
   3. Place 1.5 mL tube onto end of exit loop. Press "GO" to run the reaction. Repeat wash procedure in step 2.2.4. Reload buffer syringes A and B until they hit the driver.
   4. Repeat steps 2.2.6.2-2.2.6.3 for each desired time point. Exception: Don't load solution containing the start reagent (i.e., MgCl₂ in this case) when quenching the zero time-point control.
   5. When finished collecting samples, enter "ESC" to return to main menu. Press "7" to return buffer syringe driver to its home position.
7. Instrument clean-up
   1. Remove reaction mixture syringes. Turn sample loading knob to "LOAD" position. Under vacuum, wash each sample loop with 10 mL 0.2 N NaOH, followed by 10 mL 0.3 N H₃PO₄ and 10 mL MeOH.
   2. Turn syringe knobs to "LOAD" position. Press down on all 3 syringe plungers to retrieve reaction and quench buffers. Wash syringes with 5 mL H₂O at least twice.
   3. Fill syringes again with 5 mL H₂O and turn knobs to "FIRE" position. Change knob positions and turn on vacuum to remove H₂O. Flush as in step 2.2.4. Turn off instrument.
      NOTE: Products are analyzed as described in steps 1.7-1.12. In addition, T7 DNA polymerase and other replication proteins can be added in the reaction mixture to analyze their effect on primer synthesis or extension.

3. Single-turnover Primer Synthesis Reaction

NOTE: Single-turnover primer synthesis/extension assays are carried out similarly to multiple-turnover reactions, with a few major differences: these assays follow the conversion of radiolabeled ATP into primer or extension products, however the concentration of substrates and proteins are considerably higher. In addition, in order to attain millisecond resolution to allow analysis of the primer synthesis reaction, one must utilize a rapid-quench flow machine.

1. Preparation of reaction mixtures.
   1. Prepare 400 µL of solution A, containing 1x buffer, 3 µM gp4 hexamer, 6 µM ssDNA template, 0.6 mM dNTPs, 1 mM CTP, and 2 nM [γ-³²P] ATP. Prepare 400 µL of solution B, containing 1x buffer and 20 mM MgCl₂.
2. Rapid-quench flow operation
   1. Carry out reactions as described in step 2.2. If desired, add T7 DNA polymerase or T7 single-stranded-DNA binding protein at a concentration of 15 µM and 20 µM, respectively. In this case, the extension products accumulate at a time regime that allows for manual sampling of the reaction.

4. Data Analysis

1. Fit product progress curves to non-linear least-squares models using commercially available software capable of least-squares fits and/or numerical integration. The details of the operation will vary with the software employed, but below are generalized forms of equations used for fitting the data.
2. Determine the rate of product formation of multiple turnover primer synthesis reactions by fitting to progress curves to a linear equation. If there is significant curvature, use the initial rate method^13,14 by fitting the data to a single-exponential function: y=A(e^-kt ), where y is the product concentration, A is the reaction amplitude, k is the observed rate constant, t is time, in seconds. The slope of the tangent line at time=0 is the initial rate.
   NOTE: Fit pre-steady state primer synthesis reactions to a linear equation or to the pre-steady state burst equation y=A (e^-k_burst^t )+rate_ss t, where A is the reaction amplitude, k_burst is the observed rate constant for the pre-steady state burst, rate_ss is the steady-state rate_ss =k_cat [Enzyme]), t is time, in seconds. Fit single-turnover primer synthesis reactions progress curves to a single-exponential function y=A(e^-kt). In the case of gp4, fit the data by numerical integration¹⁵, to a model with three NTP condensation steps, where each intermediate is in equilibrium with the enzyme, using commercially available software.

Results

The results shown in Figure 1A were obtained as described in step 1 of the protocol, i.e., by manually sampling a primer synthesis reaction catalyzed by gp4 under multiple-turnover conditions. Here, a range of products after gel electrophoresis can be observed by using CTP labeled with ³²P at the α-position in primer synthesis reactions (Figure 1A). The labeled precursor, CTP, shows the highest mobility and migrat...

Discussion

The most important factor in performing these experiments is the availability of highly active purified enzyme. During our work with gp4, for example, we found that storage of the purified enzyme in buffer (20 mM potassium phosphate, pH 7.4, 0.1 mM EDTA, 1 mM DTT) containing 50% glycerol at -20 °C leads to a reduction in the specific activity of the preparation over a few months. Therefore we now flash-freeze small aliquots of purified gp4 in 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM TCEP, and 10% glycer...

Materials

Name	Company	Catalog Number	Comments
Tris pre-set crystals pH 7.5	SIGMA	T4128
Tris base	SIGMA	93362
L-Glutamic acid potassium salt monohydrate	SIGMA	G1501
Magnesium chloride hexahydrate	Mallinckrodt Chemicals	5958-04
1,4-Dithiothreitol	J.T. Baker	F780-02
Sodium Dodecyl Sulfate	MP Biomedicals	811030
(Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate	Mallinckrodt Chemicals	4931-04
[α-32P] CTP	PerkinElmer	BLU508H	radioactive, take protective measures
[γ-32P] ATP	PerkinElmer	BLU502A	radioactive, take protective measures
100 mM dATP, dCTP, dGTP, dTTP	Affymetrix	77100	four dNTPs part of set
100 mM ATP and CTP	Epicentre	RN02825	part of NTP set
ssDNA constructs	Integrated DNA Technologies	N/A	custom sequences
methanol	Macron Fine Chemicals	3017-08
formamide	Thermo Scientific	17899
bromophenol blue	SIGMA	B8026
cylene xyanol	SIGMA	X4126
acrylamide	SIGMA	A9099	Toxic
N,N′-Methylenebisacrylamide	SIGMA	M7279	Toxic
Urea	Boston Bioproducts	P-940
Boric acid	Mallinckrodt Chemicals	2549
Rapid Quench-Flow Instrument	Kintek Corp.	RQF-3
Kintek Explorer	Kintek Corp.	N/A
Kaleidagraph	Synergy Software	N/A