Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: Inherent potential for biosynthetic versus live recombinant microbicides

<p>Abstract</p> <p>Background</p> <p>Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of ~120–200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases) with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2.</p> <p>Methods and Results</p> <p>Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6%) had potential to cleave the HSV-2 genome in > 100 but < 400 sites; 69(23.9%) in > 400 but < 700 sites; and the 9(3.1%) enzymes: BmyI, Bsp1286I, Bst2UI, BstNI, BstOI, EcoRII, HgaI, MvaI, and SduI cleaved in more than 700 sites. But for the 4: PacI, PmeI, SmiI, SwaI that had no sign of activity on HSV-2 genomic DNA, all 130(45%) other enzymes cleaved < 100 times. In silico palindromics has a PPV of 99.5% for in situ REase activity (2) Two models detailing how the REase EcoRII may be applied in developing interventions against HSV-2 are presented: a nanoparticle for microbicide development and a "recombinant lactobacillus" expressing cell wall anchored receptor (truncated nectin-1) for HSV-2 plus EcoRII.</p> <p>Conclusion</p> <p>Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission.</p

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 mismatchrepair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulenceon DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broadspectrum antibiotics. Methodology/Principal Findings: herein we report the expression and purification of Yersinia pestisDam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that issuitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Daminhibitors. 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 inseparation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays weremonitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisationof Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated forhigh throughput screening, giving a Z-factor of 0.7160.07 indicating that it is a sensitive assay for the identification ofinhibitors. Conclusions/Significance: the assay is therefore suitable for high throughput screening for inhibitors of DNAadenine methyltransferases and the kinetic characterisation of the inhibitio