Title
A method to assay inhibitors of DNA polymerase IIIC activity
UMMS Affiliation
Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology
Date
4-26-2008
Document Type
Article
Medical Subject Headings
Animals; Bacillus subtilis; Bacterial Proteins; purification; Cattle; Cloning, Molecular; DNA; DNA Polymerase III; purification; DNA Primers; Enzyme Inhibitors
Disciplines
Life Sciences | Medicine and Health Sciences
Abstract
The need for new drugs to treat infections caused by antibiotic-resistant bacterial strains has prompted many studies to identify novel targets in pathogenic bacteria. Among the three DNA polymerases expressed by bacteria, one of these, designated pol III, is responsible for DNA replication and growth of bacteria and, therefore, warrants consideration as a drug target. However, the pol III enzymes of Gram-positive and Gram-negative species are quite different, and the Gram-positive enzyme pol IIIC has been more extensively studied as a drug target than the Gram-negative enzyme pol IIIE.DNA polymerases are unique enzymes with respect to the five substrates (four dNTPs, one of which is radiolabeled, and primer:template DNA) that they typically utilize. Variations of the assay, e.g., by leaving out one dNTP but allowing measurable incorporation of the remaining substrates, or use of homopolymer primer:templates, may be used to simplify the assay or to obtain mechanistic information about inhibitors. Use of gel analysis of primer extension assays can also be applied to study alternate substrates of DNA polymerases. Methods to isolate pol IIIC from Gram-positive bacterial cells and to clone and express the polC gene are described in this chapter. In addition, the assay conditions commonly used to identify and study the mechanism of inhibitors of pol IIIC are emphasized.
Rights and Permissions
Citation: Methods Mol Med. 2008;142:25-36. Link to article on publisher's site