Single molecule analysis of individual enzymes can require oriented immobilization of the subject molecules on a detection surface. As part of a technology development project for single molecule DNA sequencing, we faced the multiple challenges of immobilizing both a DNA polymerase and its DNA template together in an active, stable complex capable of highly processive DNA synthesis on a nonstic...

The inefficiency of gene modification by homologous recombination can be overcome by the introduction of a double-strand break (DSB) in the target. Engineering the endonucleases needed, however, remains a challenging task that limits widespread application of nuclease-driven gene modification. We report here that conjugates of orthophenanthroline (OP), a DNA cleaving molecule, and triplex-formi...

We describe the partial purification and characterisation of five Type II restriction endonucleases from two strains of Herpetosiphon giganteus. One of the activities, HgiJII, was the first enzyme found that cleaves DNA at the family of related sequences 5'-G-R-G-C-Y/C-3'. This enzyme may be related to the enzyme HgiAI from a different strain of the same species, and which cleaves at the sites ...

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, bu...

Recombination plays a dominant role in the evolution of the bacterial pathogen Helicobacter pylori, but its dynamics remain incompletely understood. Here we use an in vitro transformation system combined with genome sequencing to study chromosomal integration patterns after natural transformation. A single transformation cycle results in up to 21 imports, and repeated transformations generate a...

We report the characterization and cloning of the genes for an unusual type IV restriction-modification system, BspLU11III, from Bacillus sp. LU11. The system consists of two methyltransferases and one endonuclease, which also possesses methyltransferase activity. The three genes of the restriction-modification system, bsplu11IIIMa, bsplu11IIIMb and bsplu11IIIR, are closely linked and tandemly ...

Protein-DNA interactions are vital for many processes in living cells, especially transcriptional regulation and DNA modification. To further our understanding of these important processes on the microscopic level, it is necessary that theoretical models describe the macromolecular interaction energetics accurately. While several methods have been proposed, there has not been a careful comparis...

Dimeric restriction endonucleases and monomeric modification methyltransferases were long accepted as the structural paradigm for Type II restriction systems. Recent studies, however, have revealed an increasing number of apparently dimeric DNA methyltransferases. Our initial characterization of RsrI methyltransferase (M.RsrI) was consistent with the enzyme functioning as a monomer, but, subseq...

To determine relationships between Helicobacter pylori geographical origin and type II methylase activity, we examined 122 strains from various locations around the world for methylase expression. Most geographic regions possessed at least one strain resistant to digestion by each of 14 restriction endonucleases studied. Across all of the strains studied, the average number of active methylases...

This study focuses on the development of DNA catalysts (deoxyribozymes) that modify side chains of peptide substrates, with the long-term goal of achieving DNA-catalyzed covalent protein modification. We recently described several deoxyribozymes that modify tyrosine (Tyr) or serine (Ser) side chains by catalyzing their reaction with 5'-triphosphorylated RNA, forming nucleopeptide linkages. In e...