https://mmbr.asm.org/content/mmbr/48/4/273.full.pdf

Biology of bacterial deoxyribonucleic acid topoisomerases.

Publisher Summary Topoisomerases are a diverse and important group of enzymes. Although attention has until recently been focused on their ability to interconvert DNA topoisomers, this does not necessarily constitute the primary biological function for all of them. DNA topoisomerases are enzymes that catalyze changes in the topology of circular DNA. With a closed-circular double-stranded DNA, one type of reaction alters the number of times the two strands are wound around each other and thus changes the degree of supercoiling. Supercoiled DNA molecules are prevalent in cells, and enzymes that can modify this property are important in DNA metabolism. Reactions involving other topological isomers of DNA are also known; various topoisomerases can form or resolve knotted or catenated structures in circular duplex DNA, or form knots in single-stranded circular DNA. Some of these reactions also have biological importance; for instance, replication of a circular-duplex DNA often produces two catenated circles, which then have to be separated. Cells of all organisms examined to date have been found to contain DNA topoisomerases; commonly there are several distinct types in a cell. Where a genetic test has been feasible, the presence of at least one topoisomerase has been found to be essential for cell growth. This chapter discusses various lines of information bearing on the enzymatic mechanisms of topoisomerases.

Mechanistic Aspects of DNA Topoisomerases

Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination.

Integration host factor (IHF) is a small, basic protein that is needed for efficient recombination of bacteriophage lambda, as well as for other host and viral functions. We have constructed strains in which the two subunits of IHF, encoded by the himA and hip genes of Escherichia coli, are expressed under the control of the lambda rho L promoter. Separate overexpression of himA and hip led to the production of unstable and insoluble peptides, respectively. In contrast, the overexpression of both genes conjointly led to the accumulation of large amounts of active IHF. Extracts of such cells provided the starting material for a rapid purification procedure that results in milligram quantities of apparently homogeneous IHF.

Overproduction of Escherichia coli integration host factor, a protein with nonidentical subunits.

Integrative recombination of bacteriophage lambda requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation of nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg2+, spermidine, and single-stranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination.

https://www.pnas.org/content/pnas/76/8/3760.full.pdf

Nicking-closing activity associated with bacteriophage lambda int gene product.

The bacteriophage lambda int gene product. A filter assay for genetic recombination, purification of int, and specific binding to DNA.

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The bacteriophage lambda int gene product. A filter assay for genetic recombination, purification of int, and specific binding to DNA.