Model of DNA strand cleavage by topoisomerase I. Formation of the covalent bond involving tyrosine 723 is shown, as are other active site amino acids believed to function in the cleavage process.

A model for topoisomerase II action. As indicated, ATP binding to the two ATPase domains causes them to dimerize and drives the reactions shown. Because a single cycle of this reaction can occur in the presence of a non-hydrolyzable ATP analog, ATP hydrolysis is thought to be needed only to reset the enzyme for each new reaction cycle. This model is based on structural and mechanistic studies of the enzyme

Action of topoisomerases during DNA replication (A) As the two strands of template DNA unwind, the DNA ahead of the replication fork is forced to rotate in the opposite direction, causing circular molecules to become twisted around themselves. (B) This problem is solved by topoisomerases, which catalyze the reversible breakage and joining of DNA strands. The transient breaks introduced by these enzymes serve as swivels that allow the two strands of DNA to rotate freely around each other.

Structure of a Topoisomerase.      The structure of a complex between a fragment of human topoisomerase I and DNA.

Structure of Topoisomerase II.      A composite structure of topoisomerase II formed from the amino-terminal ATP-binding domain of E. coli topoisomerase II (green) and the carboxyl-terminal fragment from yeast topoisomerase II (yellow). Both units form dimeric structures as shown.

The mode of action of Type I and Type II DNA topoisomerases. (A) A Type I topoisomerase makes a nick in one strand of a DNA molecule, passes the intact strand through the nick, and reseals the gap. (B) A Type II topoisomerase makes a double-stranded break in the double helix, creating a gate through which a second segment of the helix is passed.

Topoisomerase I Mechanism. On binding to DNA, topoisomerase I cleaves one strand of the DNA through a tyrosine (Y) residue attacking a phosphate. When the strand has been cleaved, it rotates in a controlled manner around the other strand. The reaction is completed by religation of the cleaved strand. This process results in partial or complete relaxation of a supercoiled plasmid.

The reversible nicking reaction catalyzed by a eucaryotic DNA topoisomerase I enzyme. As indicated, these enzymes transiently form a single covalent bond with DNA; this allows free rotation of the DNA around the covalent backbone bonds linked to the blue phosphate.

The DNA-helix-passing reaction catalyzed by DNA topoisomerase II. Identical reactions are used to untangle DNA inside the cell. Unlike type I topoisomerases, type II enzymes use ATP hydrolysis and some of the bacterial versions can introduce superhelical tension into DNA. Type II topoisomerases are largely confined to proliferating cells in eucaryotes; partly for that reason, they have been popular targets for anticancer drugs.

Topoisomerase II Mechanism. Topoisomerase II first binds one DNA duplex termed the G (for gate) segment. The binding of ATP to the two N-terminal domains brings these two domains together. This conformational change leads to the cleavage of both strands of the G segment and the binding of an additional DNA duplex, the T segment. This T segment then moves through the break in the G segment and out the bottom of the enzyme. The hydrolysis of ATP resets the enzyme with the G segment still bound.

Model of topoisomerase 2 catalysis. The DNA duplex that undergoes cleavage is referred to as the G-segment (for “gate”) and the other DNA duplex is referred to as the T-segment (for “transported”). Binding of the G-segment (step 1) results in a conformational change (step 2) in which the active site tyrosines (shown as purple circles) are brought into position for cleavage of the G-segment. After binding of the T-segment and ATP, a “clamp” is formed around the T-segment (step 3), which is then transported though the gap in the G-segment (step 4). Subsequently, the G-strand is religated and the Tsegment is released (step 5). After ATP hydrolysis, the “clamp” is opened and the cycle can repeat

Linking Number. The relations between the linking number (Lk), twisting number (Tw), and writhing number (Wr) of a circular DNA molecule revealed schematically