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

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.

Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1)

“…1D). The pre-steady state rate was calculated as previously described, 43 providing a k obs of 0.069±0.005 min −1 . We also determined the k obs values with double-stranded (ds) DNA.…”

“…For example, histones catalyze the cleavage of AP sites in the nucleosome with k dis of 0.00036 min −1 , while human 8-oxoguanine glycosylase (hOGG1) has a slightly higher turnover number of 0.168 min −1 . 43, 49 The low turnover numbers of lyases often are due to a rate-limiting step after the chemical reaction, i.e, product release slows down the overall rate. 43, 49–52 It is often speculated that slow product release is an important feature of DNA repair enzymes in order to protect the broken DNA ends from degradation.…”

“…43, 49 The low turnover numbers of lyases often are due to a rate-limiting step after the chemical reaction, i.e, product release slows down the overall rate. 43, 49–52 It is often speculated that slow product release is an important feature of DNA repair enzymes in order to protect the broken DNA ends from degradation. In agreement with this general behavior for lyases, time course assays revealed a biphasic curve indicating that a step after strand cleavage also is rate limiting in ALKBH1.…”

Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine

“…max (KY). Human OGG1 and APE1 were purified as previously described (50,51). RNase H2 was purchased from New England Biolabs (MA).…”

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosine

“…The products were separated with 18% denaturing polyacrylamide gels (w/v) and visualized with a Typhoon system (GE Healthcare). After quantification using ImageJ software, the results were fit to a burst equation using GraphPad Prism: y ϭ A(1 -e Ϫkpt ) ϩ k ss E 0 t, where A is the burst amplitude, representing the apparent concentration of the active form of the enzyme, k p is the burst rate, k ss is the steady-state rate, and E 0 is the total enzyme concentration (30,37,40).…”