BIOC3390 Tutorials: 16 & 18 November 1999

Topoisomerases

These enzymes catalyse the transient breaking and rejoining of DNA strands. The type I enzymes cleave only one of two stands, but type II enzymes cleave both strands at the same time, allowing one DNA duplex to pass through another. Topoisomerases control the degree of supercoiling and are required for undoing knots and tangles in the genetic material. Such problems are inevitable in eukaryotic nuclei, where several metres of DNA, rotating at high speed during replication, are twisted and folded into a space only a few microns across. These enzymes are essential for DNA replication and are important targets for anti viral, anti bacterial and anti tumour drugs.

This is the X-ray structure of human topoisomerase I, reported by Redinbo et al in 1998. [Brookhaven code 1A31.] Switch to ribbon view and colour in the protein and the DNA strands. Click HERE for a brief reminder of the main CHIME commands, or HERE for a full tutorial. Rotate the molecule and experiment with different views to study the mechanism.


Topoisomerase II is is a major nuclear protein present in a large number of copies. It is associated with the nuclear scaffold, and may recognise repeated DNA motifs 30 - 90 kb apart. There are two isoenzymes of topoisomerase II (alpha and beta) which are coded on separate chromosomes. It is not clear what advantage derives from this arrangement. In bacteria this function is discharged by topoisomerase IV.

The mechanical challenge facing these enzymes is to maintain a firm grip on the two cut ends of one DNA duplex, while folding themselves around the other intact duplex as it passes through the break. Losing hold could be a disaster. Both the type I and type II enzymes proceed via a phosphotyrosine transesterification reaction. At least one ATP is hydrolysed during each catalytic cycle, probably to drive the conformational changes. It is a mechanical operation of some dexterity which justifies the designation as molecular machines.

Outline the reaction mechanism to your colleages. Are these enzymes required for transcription or just for DNA replication? Try to estimate the minimum number of cuts required to replicate (a) a prokaryotic and (b) a eukaryotic genome? Is the ATP requirement likely to be a significant burden for the cell?

Which of these enzymes allow free rotation of the cut ends?

Berger, JM et al (1996) Structure and mechanism of DNA topoisomerase II. Nature 379(6562), 225-232. [No electronic copies are available.]

Berger, JM (1998) Structure of DNA topoisomerases Biochim. Biophys. Acta 1400(1-3), 3-18. [See also the other papers in this issue. No electronic copies are available.]

Berger, JM et al (1998) Structural similarities between topoisomerases that cleave one or both DNA strands. Proc. Natl. Acad. Sci. USA 95(14), 7876-7881. [Click HERE for the HTML version, or HERE for PDF format.]

Burden, DA & Osheroff N (1998) Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim. Biophys. Acta 1400(1-3), 139-154. [See also the other papers in this issue. No electronic copies are available.]

Chen, SJ & Wang, JC (1998) Identification of active site residues in Escherichia coli DNA topoisomerase I. J. Biol. Chem. 273(11), 6050- 6056. [Click HERE for the HTML version, or HERE for PDF format.]

Couturier, M et al (1998) Bacterial death by DNA gyrase poisoning. Trends in Microbiology 6(7), 269-275. [No electronic copies are available.]

Fass, D et al (1999) Quaternary changes in topoisomerase II may direct orthogonal movement of two DNA strands. Nature Structural Biology 6(4), 322-326. [Write down the volume and page numbers for future reference. Click HERE for access instructions. Click HERE to gain access via the Leeds University network.]

Kampranis, SC et al (1999) A model for the mechanism of strand passage by DNA gyrase. Proc. Natl. Acad. Sci. USA 96(15), 8414-8419. [Click HERE for the HTML version, or HERE for PDF format.]

Levine et al (1998) DNA gyrase and topoisomerase IV: Biochemical activities, physiological roles during chromosome replication, and drug sensitivities. Biochim. Biophys. Acta 1400(1-3), 29-43. [See also the other papers in this issue. No electronic copies are available.]

Morais Cabral, JH et al (1997) Crystal structure of the breakage-reunion domain of DNA gyrase. Nature 388, 903-906. [Write down the volume and page numbers for future reference. Click HERE for access instructions. Click HERE to gain access via the Leeds University network.]

Olland, S & Wang, JC (1999) Catalysis of ATP hydrolysis by two NH2-terminal fragments of yeast DNA topoisomerase II. J. Biol. Chem. 274(31), 21688-21694. [Click HERE for the HTML version, or HERE for PDF format.]

Redinbo, MR et al (1998) Crystal Structures of Human Topoisomerase I in Covalent and Noncovalent Complexes with DNA. Science 279 1504. [No electronic copies are available.]

Stock, J (1999) Signal transduction: Gyrating protein kinases. Current Biology 9(10), R364-R367. [No electronic copies are available.]

Wang, JC (1996) DNA Topoisomerases. Ann. Rev. Biochem. 65, 635 692. [Click HERE for the abstract and citations. Unfortunately, Annual Reviews were just starting their on-line service in 1996 and there is no full-text version in PDF format. The citation list is helpful, and there are paper copies of the review in the libraries.]

Watt, PM & Hickson, ID (1994) Structure and function of type II DNA topoisomerases. Biochem. J. 303, 681-695. [No electronic copies are available.]

Most electronic journals check your browser address and require an institutional subscription to the printed version of the journal. Consequently, many of the hypertext links in this reference list will only work from a university campus computer.



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