The Active Site and Catalytic Strategy of Topoisomerase IB

This is human topoisomerase I covalently bound to DNA. The topoisomerase molecule wraps itself completely around the DNA substrate. All three subdomains of the core and the carboxy-terminal domain interact with the DNA. The interactions involving subdomains I and III are very extensive. The nose cone helices fo the cap of the enzyme offer a highly positively charged surface but the DNA does not interact directly with the DNA. Extensive protein-DNA contacts do occur is subdomain I. The protein contacts the protein contacts only the central 10 bp of DNA (from -4 to +6). There is only one base specific contact between the lysine 532 and the O-2 carbonyl oxygen of the -1 thymadine base on the scissile strand.

The catalytic tyrosine 723 is covalently attached by a phosphodiester bond to the 3' end of the scissle strand of DNA. The phosphate of the tyrosine-DNA phosphodiester bond makes close interactions with the guanidinium groups of Arg 488 and Arg 590.

The differences between the covalent and noncovalent complex were analyzed especially with regard to these residues and found that the differences are confined mainly to the catalytic tyrosine and to the scissile phosphate group. So the formation of the covalent complex does not seem to require marked structural changes in the protein or the DNA (Redinbo, 1998).

The 70-kD amino terminally truncated form of human topoisomerase I was used as a model for the mechanism of topoisomerase IB. The tyrosine 723 is positioned for nucleophilic attack and covalent attachment to the 3' end of the broken strand because it is colinear with the O5'-P scissile bond. Arg 488 interacts with the nonbridging O1, Arg 590 is also near O1, and His 632 is next to the other nonbridging oxygen, O2. The arginine 488 and 590 contribute to the stabilization though hydrogen bonding to one of the nonbridging oxygen atoms of the scissile phosphate. The His 632 stabilizes the other oxygen and might act as a proton donor to the 5' leaving group during the cleavage reaction, but no nearby base has been found.

The Reaction

Relaxation most likely occurs through a mechanism known as controlled rotation. According to this theory, the ionic interaction between the DNA and both the nose cone helices and the linker domain regulate the winding process. The nose cone helices and the linker domain are highly positively charged and may interact with the DNA during topoisomerization.

The controlled rotation mechanism involves seven steps. First, the topoisomerase must exist in an open conformation, which is most likely achieved by a hinge-bending motion located at the interface between core subdomains I and III and the boundary between helices 8 and 9. The DNA binds directed by the surface and charge complementation and the DNA is surrounded by the topoisomerase such that the lips of core subdomains I and III touch each other. This brings that active site residues in position for attack on the scissile phosphate, leading to cleavage and covalent attachment of the enzyme to the 3' end of the DNA. The release of supercoiled tension can occur through controlled rotation of the DNA. Then the DNA is released from tyrosine 723 and the whole DNA molecule is released (Stewart, 1998).

View Steps

The topoisomerase moves along the DNA strand by transiently loosing its hold on the downstream segment of DNA topological change occurs as this loose strand rotates relative to the upstream duplex. Both ends of the DNA are held by the enzyme, which then opens like a pair of jaws to let another DNA pass through the break before it is sealed (Nash, 1998).

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Updated:  December 11, 1998

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