title: 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.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, Canada, M5S 3E5.
authors.forename: Natalia
authors.initials: N
authors.lastname: Beloglazova
authors.forename: Sofia
authors.initials: S
authors.lastname: Lemak
authors.forename: Robert
authors.initials: R
authors.lastname: Flick
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
mesh_terms: D064130:CRISPR-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D045566:DNA, Cruciform / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D045566:DNA, Cruciform; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D004247:DNA; D004706:Endodeoxyribonucleases; C557650:YgbT protein, E coli