Investigation of DNA Double-Strand Breaks Induced in Host Cells Following Infection with Genotoxic Bacteria.

Abstract

Carcinogenesis is caused by genome instability, one of the major causes of which is double-strand DNA breaks (DSBs). Interestingly, infection by particular species of bacteria can induce DSBs in host cells. For example, several reports suggest an association between periodontal disease and oral cancer. Aggregatibacter actinomycetemcomitans, a common periodontal pathogen, causes DSBs in the host cell. Pulsed-field gel electrophoresis (PFGE) is often used to identify DSBs in host cells. However, as established during investigation of A. actinomycetemcomitans infection, it is often difficult to determine whether broken DNA fragments are indeed from human chromosomes or whether they are bacterial in origin using PFGE-based methods. Because the method involves the coculture of human cells with bacteria, both bacterial and human DNA fragments may be present in the broken DNA fraction. To address this problem, we have developed a method to detect only human chromosomal DNA upon PFGE analysis. Human chromosomes were prelabeled with halogenated deoxyuridine (e.g., BrdU and IdU) before being fractionated by PFGE and visualized by immunoblotting. As proof of concept, we successfully used this method to investigate the mechanism of DSB formation in host chromosomes following infection with genotoxic bacterial species.