Purification and In Vitro Analysis of Escherichia coli RecN

The RecN protein, a member of the SMC (structural maintenance of chromosomes) family, is highly conserved among bacteria and plays a critical role in the repair of DNA double-strand breaks (DSBs). Like other SMC proteins, RecN forms a ringlike structure and exhibits topological DNA-binding activity, enabling it to encircle DNA molecules. This activity is thought to be essential for maintaining nucleoid integrity at DSB sites and facilitating RecA-mediated recombination reactions. Here, we describe the purification process of the E. coli RecN protein and the assays used to evaluate its ability to mediate intermolecular DNA tethering through topological entrapment, as well as its role in facilitating strand exchange reactions. These biochemical characterizations provide insights into the function of RecN during RecA-mediated DSB repair.