Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes.

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

DNA repair enzymes of the Antarctic Dry Valley metagenome.

Microbiota inhabiting the Dry Valleys of Antarctica are subjected to multiple stressors that can damage deoxyribonucleic acid (DNA) such as desiccation, high ultraviolet light (UV) and multiple freeze-thaw cycles. To identify novel or highly-divergent DNA-processing enzymes that may enable effective DNA repair, we have sequenced metagenomes from 30 sample-sites which are part of the most extensive Antarctic biodiversity survey undertaken to date. We then used these to construct wide-ranging sequence similarity networks from protein-coding sequences and identified candidate genes involved in specialized repair processes including unique nucleases as well as a diverse range of adenosine triphosphate (ATP) -dependent DNA ligases implicated in stationary-phase DNA repair processes. In one of the first direct investigations of enzyme function from these unique samples, we have heterologously expressed and assayed a number of these enzymes, providing insight into the mechanisms that may enable resident microbes to survive these threats to their genomic integrity.