11] Escherichia coli DNA gyrase

Abstract

Gyrase's varied activities in vitro probably reflect a varied physiological role. Besides its documented place in DNA supercoiling, gyrase may also be involved in resolving catenanes and knots in the bacterial cell. Deoxyribonucleic acid gyrase is a prokaryotic DNA topoisomerase essential for cell growth that is responsible for maintaining the negatively supercoiled state of DNA. Gyrase changes the topological linking number of closed circular DNA in steps of two by transiently creating a double-strand break and passing another DNA segment through the break. When ATP is present, the result of strand passage is the introduction of negative supercoils. In the absence of ATP, gyrase removes negative supercoils, albeit inefficiently. The Escherichia coli gyrase, which is discussed in this chapter, has been best characterized, but the Micrococcus luteus enzyme is strikingly similar in spite of the evolutionary divergence of these bacteria. The availability of selective inhibitors and mutants has allowed extensive physiological studies of gyrase's role in vivo and determination of the particular function of each subunit. The chapter discusses the assay, purification, and reactions of DNA gyrase. The enzyme is a valuable reagent for preparing negatively and positively supercoiled DNA, forming or resolving catenanes and knots, and analyzing the structure of complex forms of DNA.