The use of alkaline gel electrphoresis to analyze hydrogen peroxide-caused DNA damage and repair in Escherichia coli

Abstract

Reactive forms of oxygen such as hydrogen peroxide cause single-strand breaks in DNA. Most of the methods for estimating such breakage and subsequent repair are designed for eucaryotic cells and methods for use with bacteria are needed. Accordingly, a method based on alkaline gel electrophoresis of DNA was developed and tested with isogenic strains of Escherichia coli deficient in one or more DNA repair enzymes, viz., recA (recA), exonuclease III (xthA), DNA polymerase I (po/A) or DNA polymerase I plus exonuclease I] (polA-xthA). For DNA analysis of single-strand breaks, samples from a cell suspension were removed at 2 min intervals following an initial 15 min exposure to 20 mmol l⁻¹ hydrogen peroxide. Catalase was added and the cells were embedded in blocks of low-melting point agarose and lysed to liberate their DNA . After alkaline gel electrophoresis, photographs of the gels were taken and the lengths of the distributions of DNA fragments were measured with a scanner and computer. The wild type and recA strain showed only a moderate increase in the length of the DNA distribution whereas the remaining strains all showed a large increase in the length of the distributions. The lengths of the distributions were correlated with cell survival at the same concentration of H₂O₂ and with the importance of particular DNA repair enzymes. Alkaline gel electrophoresis appears to be a relatively simple method for analyzing the level of H₂O₂-caused DNA damage and repair in E. coli.