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 ....

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 ....

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 migrates to.......

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

List of materials used in this article

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