Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide

Abstract

Background: DNA 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 findings: Herein 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/significance: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

Figure 1. Break light Dam activity assay.

The fluorescence of the hemimethylated substrate oligonucleotide 1 is internally quenched by the dabcyl group. It is a substrate for Dam and yields the fully methylated product 2, which is rapidly cleaved by DpnI, thus forming fluorescent oligonucleotide 3.

Figure 2. Activity of Y. pestis Dam with hemimethylated oligonucleotide substrate.

The fluorescence increase was recorded with Dam at the following concentrations: 0 nM (dotted line), 0.31 nM (dashed line) and 0.61nM (solid line).

Figure 3. Inactivation of Y. pestis Dam.

Dam was incubated in a PCR machine at 30 °C with the hemimethylated substrate oligonucleotide (triangles), with AdoMet (open circles) or with no additions (filled circles). Aliquots were withdrawn at time intervals and the activity measured. Activity is expressed as a percentage of the initial activity.

Figure 4. Dependence of Dam activity on substrate concentration. (A) AdoMet and (B) oligonucleotide 1.

Figure 5. Inhibition of Dam by S-adenosylhomocysteine.

(A) A double reciprocal plot of methylation rate against AdoMet concentration. S-adenosylhomocysteine concentrations were; 0 (•), 5 (○), 10 (▾), 15 (Δ), 20 (▪), 30 (□) and 40 (♦) µM. (B) Apparent K _M plotted against the concentration of S-adenosylhomocysteine.

Figure 6. High throughput assay validation.

Rate of fluorescence increase was monitored over 96 wells (wells 1–48 contained positive control assays, wells 49–96 contained negative control assays with no Dam). The solid trend lines indicate the mean rate of fluorescence increase for the positive/negative control datasets; the broken lines indicate the ±3 standard deviations from the mean, which is the 99.7% confidence limit.