Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Summary

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Abstract

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, k_off). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (k_off). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (<1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E>S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Introduction

An aerobic environment hastens genomic instability. A major promutagenic DNA lesion resulting from oxidative stress is 7,8-dihydro-8-oxoguanine (8-oxoG). This is due to the ambiguous coding potential of 8-oxoG. Human 8-oxoguanine DNA glycosylase (OGG1) is responsible for initiating base excision repair of 8-oxoG. The glycosylase activity of OGG1 excises the 8-oxoG base resulting in product DNA with an apurinic site (AP-site). A weak lyase activity of OGG1 can incise the AP-site in some instances.

Kinetic characterization of DNA glycosylases generally finds that they exhibit biphasic time courses. After an initial fast phase....

Protocol

1. Preparation of Enzyme and DNA Substrate

1. Over-express OGG1 as a GST-fusion protein in E. coli, utilize the GST-tag for purification, and then remove the GST-tag by cleavage with HRV-3C protease (Figure 1)⁶.
2. Purchase the chemically synthesized 5'-6-carboxyfluorescein (6-FAM) labeled oligonucleotide containing a single 8-oxoG residue and its complementary unlabeled oligonucleotide strand. The 34-mer oligonucleotides contain an 8-oxoG at position 17 from the 5'-end. Purify these oligonucleotides by polyacrylamide gel electrophoresis.
3. Prepare double-stranded DNA substrate containing 8-oxoG by mixing 5'....

Results

Steady-state kinetic analysis was performed by using 200 nM DNA substrate and four different apparent concentrations of OGG1 (15, 30, 45, and 60 nM) as determined by a Bradford protein assay². The time courses of product formation were fit to a linear equation to determine the y-intercept, which were 2.2, 11, 15, and 26 nM, respectively, relative to each protein concentration (Figure 2B). The y-intercepts were further plotted relative to each actual protein concentration (Figure 2C

Discussion

The kinetic approaches described here outline methods to define elementary kinetic constants. If a time course of product formation is biphasic with the first enzymatic turnover occurring rapidly, then a step after chemistry is rate-limiting during subsequent catalytic turnovers. In the case of OGG1, the first turnover can be measured using high enzyme concentrations with either limiting (SE) DNA concentrations. In the first case, the reaction is limited to a 'single-turnover' and provides a measure .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent/Material
5’-6-FAM labeled oligonucleotides containing a single 8-oxoG	Eurofins MWG Operon		5’-P32 radiolabeled oligonucleotides can be used as well. Polyacrylamide gel purification grade is recommended.
Unlabeled oligonucleotides (complementary strand)	Eurofins MWG Operon		Polyacrylamide gel purification grade is recommended.
1 ml BD Luer Lock disposable syringe	BD Medical	309628	Lue Lock disposable syringe from other vendors can be used as well.
10 ml BD Luer Lock disposable syringe	BE Medical	309604	Luer Lock disposable syringe from other vendors can be used as well.
Equipment
Circulating water bath	Any vender
RQF-3 Rapid Quench-Flow Instrument	KinTek Corporation		Rapid Quench-Flow Instrument from other vendors can be used as well.
Typhoon Phosphorimager 8600	GE Healthcare Life Sciences		Imager from other vendors can be used as well.
KaleidaGraph	Synergy Software