Topoisomerase is a general name encompassing a class of enzymes that catalyzes the uncoiling and supercoiling of DNA. They function by relieving the topological strain associated with the common cellular process of DNA transcription, replication, and recombination by temporarily cleaving the DNA. During the processes of transcription, the double stranded helical structure of DNA combined with its separation by the enzyme helicase, causes DNA to become supercoiled. In order for the helicase to continue unwinding the DNA, this tension must be relieved by topoisomerases. During replication, the chromosomes must be untangled in order for mitosis to successfully continue, this process is also carried about by topoisomerases. Furthermore, the overall large size of the eukaryotic genome requires the use of histones to effectively package the DNA in the nucleus. This packing of the DNA onto the histones, and interactions with other required proteins induces further supercoiling that must be relieved by topoisomerases. Cleavage of the DNA by all classes of topoisomerases utilizes a transesterification reaction in which a tyrosine hydroxyl forms a covalent, temporary linking of the enzyme to the phosphate break in the DNA backbone while an active site tyrosine is responsible for the covalent bond to the 5’ phosphate at the end of the cleaved strand. The breaking and rejoining of the strands requires no energy input since the bond energy is conserved in the protein-DNA intermediate. Topoisomerases are classified into two groups based on their mechanism of DNA cleavage.
Type I: Type I topoisomerases work by temporarily cleaving a single strand of the DNA before relegating the bond in an ATP independent manner. They are classified into two subfamilies (IA and IB). The IA subfamily includes Eubacterial DNA topoisomerase I, topoisomerase III, reverse gyrase, mammalian topoisomerase IIIα, β along with topoisomerase III found in yeast. The IB subfamily of topoisomerases includes prokaryotic topoisomerase V, poxvirus topoisomerase, and mammalian topoisomerase I.
Mammalian Topoisomerase I: Mammalian topoisomerase I is a 91 kDa enzyme with a monomer sub structure. Crystal structure work on the enzyme has led to the belief that it relaxes DNA though a more passive approach of allowing “controlled rotation” of the DNA rather than a bridging mechanism of the IA subfamily. While the exact number of rotations is not known for mammalian topoisomerase I, an average of 5 rotations has observed in poxvirus topoisomerases. It can relax both positive and negative supercoiled DNA and unlike it IA counterparts, its cleaving can occur in a double stranded section where Type IA topoisomerases requires single stranded sections. Similar to other topoisomerase I enzymes, the structure itself consists of 4 sections: The N-terminal domain (~24 kDa), core domain (~54 kDa), linker domain (~3 kDa), and C-terminal domain (~10 kDa). Together these 4 domains manifest into a hinged structure that forms a clamp around the DNA creating binding core of 15-20 Angstroms. Within the core, 15 lysines and 8 arginines create a highly positive electrostatic potential responsible for interacting with, and stabilizing, the DNA. The core itself, when clamped around the DNA, covers approximately 22-25 base pairs.
Type II: Type II topoisomerases function by utilizing Mg2+ and ATP to pull one strand (termed the Transport segment or T-segment for short) through a temporary gate created by the cleavage of the same or another DNA molecule (Gate segment or Gsegment). Like the Type I counterparts, type II topoisomerases also contain two subfamilies: IIA and IIB. Type IIA includes Eubacterial DNA gyrase, topoisomerase IV, Yeast DNA topoisomerase II, and Mammalian DNA topoisomerase IIα/IIβ while the IIB subfamily contains Archaeal DNA topoisomerase VI.
Mammalian topoisomerase II: Mammalian topoisomerase II is a 170 kDa enzyme. It prefers binding to curved DNA sections or DNA crossovers and is capable of relaxing both positive and negative supercoiled DNA. Its structure is composed of three domains: The ATPase domain, the DNA Binding/Cleavage domain, and the C-terminal tail that together fold to create a hinged protein. The arrangement of the three domains creates two internal pockets that are thought to store the DNA segment during the reaction. The ATPase domain has two ATP binding sites to facilitate the efficient relaxing of the DNA, and it has been shown that the IIα can function less efficiently with only one ATP binding site. Topoisomerase II works through a two gate mechanism. In this method, the uncut DNA strand enters the enzyme on one side and passes through the cut DNA strand before being released from the enzyme on the opposite side it came in on. This mechanical movement of the DNA through the cleaved strand and enzyme is what requires the input of ATP.
Small molecule inhibitors targeting both types of topoisomerase are commonly used in chemotherapy. As stated earlier, cellular effects of anthracyclines are mediated in part by inhibiting topoisomerase II, whereas quinoline based alkaloids such as Camptothecin target topoisomerase I. Known topoisomerase inhibitors tend to target either topoisomerase I or II, while the ability for a compound to target both topoisomerase I and II is rare among current known inhibitors. Topoisomerase inhibitors fall into three classes based on the mechanism through which they achieve their inhibition: 1) Topoisomerase poisons inhibit through stabilizing the transient break created during the cleavage/re-ligation step. They bind to the DNA, enzyme, or both, and in doing so slow down the rate at which the enzyme can relegate the DNA, or cause an increase in strand breaks by preventing re-ligation altogether. They can target either enzyme bound or free DNA and tend to be planar in nature which allows the compound to insert between DNA base pairs. While most anthracyclines bind DNA very tightly through this mechanism known as intercalation, some compounds such as Hoechst 33358 actually bind to the minor groove instead of sliding between the base pairs. Other poisons such as Camptothecin and etoposide bind to the enzyme-DNA complex formed during the cleavage step preventing relegation: 2) Catalytic inhibitors work non-covalently and unlike poisons which target the ligation step, catalytic inhibitors prevent the initial cleavage step. Inhibition by catalytic inhibitors is achieved through substrate competition (i.e. competitive binding to the ATP site in topoisomerase II: 3) The final class are compounds that are non-specific towards topoisomerase but nonetheless inhibit the enzyme through covalent modifications. One example of this is N-ethylmaleimide which has been shown to covalently bind to topoisomerase I and inactive the enzyme