1A41 | pdb_00001a41

DNA topoisomerase IB (TopIB) is an enzyme that acts to relax both negative and positive supercoils generated during transcription and DNA replication. Due to of the size of the eukaryotic chromosome, removal of these supercoils can only be accomplished locally by introducing breaks into the DNA helix. TopIB mediates DNA relaxation by creating a transient single-strand break in the DNA duplex. This transient nick allows the broken strand to rotate around its intact complement, effectively removing local supercoils. Strand nicking results from the transesterification of an active-site tyrosine (Tyr-723 in human, Tyr-274 in the viral protein) at a DNA phosphodiester bond forming a 3-phosphotyrosine covalent enzyme-DNA complex. After DNA relaxation, the covalent intermediate is reversed when the released 5-OH of the broken strand reattacks the phosphotyrosine intermediate in a second transesterification reaction. The rate of religation is normally much faster than the rate of cleavage, and this ensures that the steady-state concentration of the covalent 3-phosphotyrosyl TopIB DNA complex remains low. A variety of DNA lesions and drugs have been shown to stabilize the covalent 3-phosphotyrosyl intermediate.

Unlike most TopIBs, the enzyme isolated from the vaccinia virus exhibits specificity for cleavage at a consensus sequence: 5'-(T/C)CCTT-3' in the scissile strand with cleavage occuring after the last base. The viral TopIB is also the smallest topoisomerase known and is unusual in that it is resistant to the potent chemotherapeutic agent camptothecin (CPT). CPT is a natural product that was originally discovered because of its antitumor activity and was later demonstrated to cause the accumulation of TopIB DNA adducts in vitro and in vivo. CPTs bind the covalent 3-phosphotyrosyl intermediate and specifically block DNA religation, thus converting TopIB into a DNA-damaging agent. TopIB is the sole intramolecular target of CPT, and the cytotoxic effects of CPT poisoning are S-phase specific. During DNA replication, the replication fork is thought to collide with the trapped TopIB DNA complexes, resulting in double-strand breaks and ultimately cell death.