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

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5'-CG-3' site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 +/- 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R(2)) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5'-CG-3' site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors.

Direct and continuous fluorescence-based measurements of Pyrococcus horikoshii DNA N-6 adenine methyltransferase activity.

N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmols(-1) in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase.

Recombinant antigens based on toxins A and B of Clostridium difficile that evoke a potent toxin-neutralising immune response.

Infection with the bacterium Clostridium difficile causes symptoms ranging from mild to severe diarrhoea with life-threatening complications and remains a significant burden to healthcare systems throughout the developed world. Two potent cytotoxins, TcdA and TcdB are the prime mediators of the syndrome and rapid neutralisation of these would afford significant benefits in disease management. In the present study, a broad range of non-toxic, recombinant fragments derived from TcdA and TcdB were designed for soluble expression in E. coli and assessed for their capacity to generate a potent toxin-neutralising immune response as assessed by cell-based assays. Significant differences between the efficacies of isolated TcdA and TcdB regions with respect to inducing a neutralising immune response were observed. While the C-terminal repeat regions played the principal role in generating neutralising antibodies to TcdA, in the case of TcdB, the central region domains dominated the neutralising immune response. For both TcdA and TcdB, fragments which comprised domains from both the central and C-terminal repeat region of the toxins were found to induce the most potent neutralising immune responses. Generated antibodies neutralised toxins produced by a range of C. difficile isolates including ribotype 027 and 078 strains. Passive immunisation of hamsters with a combination of antibodies to TcdA and TcdB fragments afforded complete protection from severe CDI induced by a challenge of bacterial spores. The results of the study are discussed with respect to the development of a cost effective immunotherapeutic approach for the management of C. difficile infection.

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

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.