Kinetic Analysis of Yersinia pestis DNA Adenine Methyltransferase Activity Using a Hemimethylated Molecular Break Light Oligonucleotide

Abstract

BackgroundDNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics.Methodology/Principal FindingsHerein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71±0.07 indicating that it is a sensitive assay for the identification of inhibitors.Conclusions/SignificanceThe assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity

“…Cleavage of the stem leads to the melting of the short cleaved sequence and separation of fluorophore and quencher resulting in an increase in fluorescence. This assay format has been applied to DNA adenine-N6 methyltransferase activity (Dam) ( 38–40 ), in all cases the oligonucleotide stem contains the 5′-GATC-3′ Dam recognition sequence. The most recent assay coupled a hemimethylated substrate with a restriction enzyme (DpnI), which will preferentially cleave the fully methylated substrate, so that one methylation event by Dam is reported by a proportional increase in fluorescence because of DpnI cleavage.…”

Inhibition of Yersinia pestisDNA adenine methyltransferase in vitro by a stibonic acid compound: identification of a potential novel class of antimicrobial agents

“…Such competition binding assays took up to 1 h to complete, and the ‐term stability of Dam during these experiments was therefore an important consideration. Kinetic analysis of Y. pestis Dam inactivation has shown the enzyme to be particularly unstable (Wood et al ., ) and the more stable E. coli enzyme was used for these measurements. Both compounds 3 and 13 were found to fully displace oligonucleotide 4 in the presence and absence of AdoHcy, resulting in a drop in fluorescence anisotropy values to that of free‐labelled 4 in solution.…”

“…Fluorescence measurements were recorded in a Tecan Safire II (Reading, UK) microplate reader using 10 readings per well (each measurement) a Z-position of 12 000 mM and an integration time of 20 ms. Fluorescence measurements were made using an excitation wavelength of 486 nM with a bandwidth of 5 nM, and an emission wavelength of 518 nM with a bandwidth of 10 nM and the gain was set at 170, unless otherwise stated. Calibration plots were prepared as described previously (Wood et al, 2007) and in Supplementary Section S2 to enable conversion of rate of change in fluorescence to change of rate of reaction ( Supplementary Table S1 and Figure S1). The activity of Y. pestis Dam was measured in triplicate (unless otherwise stated) in black, flat bottomed, 384 Well Small Volume TM HiBase polystyrene microplates (Greiner, Stonehouse, UK), with a total assay volume of 20 mL, maintained at 30°C.…”

“…Both these mutants were attenuated in mouse infection models. In order to facilitate the development of Dam-specific inhibitors, we developed a continuous fluorescence-based assay for monitoring Dam activity that is suitable for the high-throughput screening (HTS) of potential Dam inhibitors (Wood et al, 2007). In this current study, we describe the screening of an inhibitor library and identification of compounds with specific activity against Dam in this assay, providing a lead for the development of antimicrobials against this target.…”

Nanopore-Based, Label-Free, and Real-Time Monitoring Assay for DNA Methyltransferase Activity and Inhibition

“…Most of these approaches are discontinuous and laborious. For real-time monitoring of DNA methyltransferase activity fluorescence-based assays based on either fluorescence quenching or fluorescence resonance energy transfer (FRET) have been proposed. − However, the proposed fluorescence sensors require double-labeled oligonucleotide substrates which will not only increase the cost of sensing system but also probably reduce the cleavage efficiency, and these bulky fluorescent groups might interfere with the kinetic behavior of the substrate molecules. Fluorescent assays require the proper design of DNA probes which may limit the versatility of such methods.…”