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 0.7160.07 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

More information

Identifiers

ORCID for Peter L. Roach: orcid.org/0000-0001-9880-2877

Catalogue record

Export record

Altmetrics