Analysis of nuclease activity of Cas1 proteins against complex DNA substrates

Abstract

Cas1 genes encode the signature protein of the CRISPR/Cas system, which is present in all CRISPR-containing organisms. Recently, Cas1 proteins (together with Cas2) have been shown to be essential for the formation of new spacers in Escherichia coli, and purified Cas1 proteins from Pseudomonas aeruginosa and E. coli have been shown to possess a metal-dependent endonuclease activity. Here we describe the protocols for the analysis of nuclease activity of purified Cas1 proteins against various DNA substrates including Holliday junctions and other intermediates of DNA recombination and repair.