Topoisomerase IB is an enzyme necessary for the successful replication, translation, and modification of DNA in Eukaryotic organisms. More recently, Topoisomerase IB has been found to be present in some species of archaebacteria which testifies to the presence of this enzyme in the last common ancestor of Archaea and Eukarya - further testifying to indispensability. 1) The necessity of this topoisomerase is due primarily to its role in the relaxation of the supercoils that form when DNA is being unwound or stored. A DNA supercoil is created when torsional stress induces the axis of the DNA double helix to bend back upon itself (Figure 2). In layman’s terms this could be described as a “coil of a coil.” Depending on the type of torsional stress applied to DNA it will become either negatively or positively supercoiled. The difference between these two states lies in the direction in which the DNA is twisted, resulting in the DNA folding over itself in opposite directions (Figure 3). Because other enzymes that are essential for cell life, such as helicase, cannot act on supercoiled DNA, it must be unwound to its relaxed state in order for these other enzymes to perform their functions. One distinguishing factor between Topoisomerase IB and Topoisomerase IA is that IB has the ability to unwind both positive and negative supercoils while IA can only accept negatively supercoiled DNA as a substrate. 2)
The catalytic action of Topoisomerase IB is characterized by the cleavage of one side of the DNA backbone, followed by a controlled ratchet-like rotation mechanism that unwinds the DNA supercoil. Structurally, the enzyme is composed of a single-unit protein comprised of four domains. Its active site architecture can be described as a “pac-man” like clamp, which tightly binds to DNA prior to cleavage and relaxation. While this “pac-man” motif is common among enzymes that interact with DNA, several aspects of its architecture are unique to the enzyme. In fact, large portions of Topoisomerase IB’s structure remains uncharacterized, especially its disorganized N-terminal domain. Research continues to be done on this enzyme, as many believe that it represents a promising target for developing potential cancer treatments. 3)
Topoisomerase IB is a single unit protein that consists of two distinct lobes: the C-terminal lobe and N-terminal lobe. However, little structural information is known about the N-terminal portion of the enzyme due to its disorganized, non-conserved structure, which is largely unnecessary for enzymatic function. In eukaryotes, the number of amino acid residues that make up the enzyme ranges from 765 to 1019 amino acids, however, even from evolutionarily distant species the structure is homologous enough to consider it one type of enzyme. The primary reason for the difference in the length of the amino acid sequences among eukaryotic species is mutations in the N-terminal lobe, which is not involved in the enzyme's primary catalytic action; in comparison, the C-terminal lobe is much more highly conserved among species. 4) Although the mass of the entire enzyme has not been determined, the C-terminal lobe has been truncated and found to have a mass of 70kDa. 5)
Three domains make up the C-terminal lobe: the linker, core, and C-terminal domains. In contrast, the entirety of the N-terminal lobe is comprised of its one N-terminal domain. Crystal characterizations are only available for the catalytic C-terminal lobe of Topoisomerase IB, and thus detailed descriptions of structure must be limited to this segment. 6) The overall architecture of the C-terminal lobe is defined by a clamp design consisting of two separate lips joined by a hinge motif – features common to many enzymes that interact with DNA. Nonetheless, Topoisomerase IB’s structure contains unique design features such as the asymmetry of its two lip structures. The unevenness of these two lips is caused by the extended helices of the linker domain on the lower lip as compared to the shorter helices present on the upper lip of C-terminal lobe (Figure 4b). Both pairs of these helices flank the DNA as it is catalytically unwound after cleavage. Yet another unique feature of the enzyme is the concentration of its catalytic residues in the central pore of the C-terminal lobe, which lends the enzyme an ability to pack the DNA duplex more tightly into its active site. 7)
The N-terminal domain is on average 210 amino acid residues in length and structurally disorganized. While the specific amino acid sequence and structure of the domain has been found to be highly non-conserved across species, several properties are found to be consistent. First, the domain is highly charged, with very few hydrophobic residues found in its sequence. While its charged nature does not play a significant role in its interaction with binding DNA, some studies have suggested that it is important in mediating other protein-protein interactions in which the N-terminal domain contains a consensus site for covalent modification by a modifying protein. 8) Another similarity found across the N-terminal domains of topoisomerase IB is the presence of Nuclear Localization Signal (NLS) motifs. These signals play a role in ensuring that the enzyme is located in the correct portion of the cell’s nucleus, an important function in coordinating its activities with translation, replication, and modification of DNA. So far, four NLS motifs have been identified through comparing homologous portions of the domain between different species’ enzymes. 9)
The Core Domain is the largest component of the globular C-terminal lobe and is divided into three sub-domains:
Sub-domain I of is comprised of residues 215-232 as well as 320-433 and consists of 3 alpha helices and 9 beta helices. Sub-domain II is comprised of residues 233-319 and consists of 10 alpha helices and 2 beta helices. 10) These two sub-domains form a cap positioned onto the DNA duplex (forming the upper lip of the “pac-man” complex). They contain alpha helices 5 and 6, which form a “nose-cone” that sits close to the bound DNA, locking it tightly in place. This “nose-cone” contains six positively charged residues that point towards the DNA duplex, however, of these residues only Arg316 makes direct contact with the nucleotides on the cleaved DNA strand (between nucleotides 15 and 16). 11) Sub-domain I connects the upper lip to Sub-domain III (on the lower lip) via a short hinge helix – so named because it is the axis around which the lips open and close during plectonemic unwinding. 12)
Sub-domain III is consists of residues 424-635 and is made up of 10 alpha helices and 3 beta helices. It makes up part of the lower lip and contains all of the catalytic amino acids involved in the active site with the exception of Tyr723.
The C-terminal domain is made up of residues 713-765 and organized into five short alpha helices. The main importance of this domain is that it contains the residue Tyr723 which acts as the nucleophile during the attack on the phospodiester bond of the DNA backbone during catalyis. This residue is 100% conserved among Topoisomerase IB enzymes.
The Linker domain is composed of the region containing residues 636-712. It gained its moniker due to its function in connecting the core domains to the C-terminal domain. Residues Asn711 and Leu716 accomplish linkage to the C-terminal domain while Pro636 and Phe640 form the bridge to the core domains. The primary structure consists of two extensive alpha helices connected by a short turn formed by residues Met675 and Ala678, creating an anti-parallel helix-twist-helix structure that lies roughly parallel to the DNA axis. The two alpha helices that comprise the domain interact extensively with each other, primarily through the hydrophobic interactions of Leucine residues (Figure 7). The top surface of the coiled-coil linker domain is strongly positively charged, as this is the area closest to the active site that interacts with DNA. Nonetheless, the domain is discontinuous with the enzyme’s active site, with the tip of the linker group 35 Angstroms distant from the nearest phosphate group of the bound DNA. Thus, the only charged residues to make direct contacts with the bound DNA are Lys650 and Arg708, which are located proximal to the core domains and are conserved across all type IB topoisomerases. The other charged residues are thought to a play a role in stabilizing the DNA as it is unwound but this has yet to be confirmed (Figure 6).13)
The C-terminal and Core domains form the primary active site of the enzyme and contain the catalytic residues necessary for the cleavage and re-ligation of the DNA backbone. In its non-active state, this active site is in an open conformation, however, upon binding to DNA, the two lips of the enzyme are clamped together. In this closed conformation, the DNA binding pocket is approximately 15-20 Angstroms in diameter. This central cavity has a high concentration of positively charged amino acid residues and a strongly positive electrical potential. This positive potential assists in tightly packing the negatively charged DNA into the active site.
Upon binding DNA, the first step in plectonemic unwinding is cleaving one of the DNA backbones. Arg590 complexes with a water molecule in the active site, which acts as a base and deprotonates Tyr723. Upon deprotonation, Tyr723’s hydroxyl oxygen becomes nucleophilic and attacks the electrophilic phosphorus on the DNA backbone – forming a covalent phosphotyrosine bond that remains in place as the DNA is unwound. The cleavage of the O5-P scissile bond is theorized to be stabilized by the hydroxyl capping mechanism of His632, which donates a Hydrogen to the cleaved Oxygen on the DNA backbone. The mechanism for the religation of DNA still remains unclear. Nonetheless it has been hypothesized that the 5’ –OH group on the cleaved DNA is deprotonated by His632 (acting as a base) and nucleophilically attacks the phosphotyrosine bond. However, this mechanism cannot be confirmed as it would require a 180 degree rotation of DNA for the 5’ –OH group to act effectively as a nucleophile and such a rotation has not been observed during religation. 14)
Despite considerable research, the exact mechanism for the unbraiding of DNA supercoils after formation of the covalent complex and before religation is unknown. The most prevalent theory is that the 5’ terminus of the broken strand is released from the active site and allowed to rotate freely around the complementary unbroken strand. According to this theory, multiple unwinding events can occur for each cleavage-religation cycle (see Figure 11). However, it would be sterically impossible for the DNA strand upstream from the cut to rotate around the DNA’s helical axis because it is so tightly bound into active site. Instead, it is theorized to rotate off-centrically around an axis parallel to the double helical axis and around the periphery of the upstream DNA duplex (Figure 9).15)
The catalytic unwinding of supercoiled DNA by Topisomerase IB does not require any input of energy by the cell. Instead, the energy is supplied by the release of torsional stress upon cleavage of one of the DNA backbones. The energy profiles for unwinding positively and negatively supercoiled DNA are very similar although it is slightly more energetically favorable to unwind a negatively supercoiled strand of DNA (Figure 11 shows the free energy profiles for both types of supercoiled DNA). Furthermore, when unwinding negatively supercoiled DNA, the hinge motif of Topoisomerase IB is flexed/stretched while the conformational change associated with unwinding positively supercoiled DNA is the opening of the active site's two lips. While it is unclear why two different conformations are utilized in unwinding positive and negative supercoils, both conformational changes serve to relax the enzyme’s hold on the DNA as it is unwound. 16)