Background: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch<br/>repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence<br/>on DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broad<br/>spectrum antibiotics. <br/><br/>Methodology/Principal Findings: herein we report the expression and purification of Yersinia pestis<br/>Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is<br/>suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam<br/>inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site.<br/>When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in<br/>separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were<br/>monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation<br/>of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for<br/>high throughput screening, giving a Z-factor of 0.7160.07 indicating that it is a sensitive assay for the identification of<br/>inhibitors. <br/><br/>Conclusions/Significance: the assay is therefore suitable for high throughput screening for inhibitors of DNA<br/>adenine methyltransferases and the kinetic characterisation of the inhibitio
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