Dioxiribose nucleic acid molecules, or
DNA, are absolutely essential to the function of all life forms on
earth. To contain the massive amount of
information held by their atomic structures, cellular molecules of DNA are
supercoiled to conserve space. This allows a collection of structures that would be two
meters long if stretched end to end to exist neatly within the confines of a cell, existing in a space a
million times smaller (Goodsell 1). The disadvantage of the supercoiling structure is that it makes the information contained within the DNA impossible to access when the time comes to read, copy, or
concatenate strands of DNA (Champoux 1). It also puts strain on the molecules, gradually building up tensions which can be
detrimental to the continued accurate replication and storage of genetic information. This problem is solved by the DNA
Topoisomerase enzymes. They are vital to the access and propagation of genetic information through the use of their unique molecular structures. The binding properties and folding of type I and II topoisomerase allow them to sever and reconnect strands of DNA to serve a wide variety of biological
purposes.
DNA Topoisomerase I is a polypeptide enzyme coded by the Topoisomerase I gene, abbreviated as TOP 1 (Flybase 1). It is found in both prokaryotic and eukaryotic cells. The structural and functional data of Topoisomerase I was obtained through crystallography studies. (Andoh, Preface). The amino acids which compose the polypeptide chain number 972 (Flybase 1). The primary structure is intricately folded by the means of secondary hydrogen bonding, extensively coiling the polypeptide chain through bonds between amino acids. These bonds usually take place every third amino acid. The tertiary structure of DNA Topoisomerase I is also vital to its function. Interactions between R-Groups of the amino acids bend the enzyme into a weighted ring shape. The inner portion of the ring is lined with basic residues. The Topoisomerase I enzyme is divided into four major domains. "Domain I is made up of a beta sheet with four parallel beta strands and four alpha helices. This domain resembles the Rossman fold known to bind nucleotides in many other proteins, and so it is a likely candidate for binding a segment of DNA. Domain II consists of two beta sheets, each with three antiparallel strands, and one alpha helix. Domain III consists of five alpha helices, and this domain also contains the active site, Tyr 319. Domain IV has eight alpha helices and apparently provides structural support for the protein" (Kysela 1). The tyrosine active site is the most important portion of the Topoisomerase molecule.
DNA Topoisomerase I solves the topological conundrums of supercoiled DNA by severing a single portion of its strand with the active Tyrosyl site of the topoisomerase. DNA is attracted into the central ring of Topoisomerase I through electrostatic interaction between the basic residues of its inner surface and the negatively charged portions of the phosphate backbones of the supercoiled DNA (Kysela 1). The negative charge of the DNA is derived from its single oxygen anion connected to each phosphate linkage. The tyrosine active site stemming from the C-Terminal group of Topoisomerase I is involved with the relaxation of positive and negative DNA supercoiling (Kysela 1). The OH group of the tyrosine active site engages in a reaction with a molecule of the DNA’s phosphor backbone. It sheds its proton and bonds the free oxygen to the phosphate atom of the backbone. The hydrogen, in turn, bonds to the now free oxygen bonded to the DNA chain itself. The end result of this reaction is the severing of the phosphate linkage that serves as one side of the DNA's backbone and the freeing of one side of the DNA's double helix (Wang 2). From this point, the DNA molecule is allowed to rotate about its one fixed axis. This freedom unravels its supercoiling, the forces of which are no longer held in place. In this way, the DNA molecule can then by read or copied, with access to its information no longer barred by its coiled structure. It also relaxes tensions caused by the process of DNA supercoiling, preventing breakage over time from wear and tear. Once the tension of the DNA has been relaxed, the phosphate backbone will naturally break the covalent bond between the tyrosine residue and phosphate, reversing the reaction previously used to bond the two and returning the rigid structure of the DNA’s backbone to its original form. The actual chemical process for the breakage of a DNA strand’s phosphate backbone occurs through the breakage of the non-covalent interfaces along the Topoisomerase I's surface. Domains II and III of the molecule swing away once the OH group of the Tyrosine residue is loosened, opening a wide gate through which the DNA strand can pass through (Kysela 1). At the end of the cycle, the nearby presence of the phosphate backbone’s original linkage severs the tenuous covalent bond between the tyrosine residue and the phosphate's oxygen. This rejoins the backbone and releases the DNA with supercoiled tension abated. Covalent bond dispersed, Domains II and III swing back to their original position and resume their non-covalent interaction that maintains DNA Topoisomerase I in its non-active form. The vast majority of biological examples of DNA Topoisomerase I show that the enzyme only reduces positive or negative supercoiling, but in specialized cases it can sometimes introduce positive supercoiling, increasing the tension within the DNA molecule instead of decreasing it (Fox, 38).
The structure of DNA Topoisomerase II shares similarities with its counterpart, DNA Topoisomerase I. It shares a polypeptide structure made up of amino acids. The number extends to 1447, which makes it more complicated than DNA Topoisomerase I (Flybase 1). The structure of this molecule is less clearly understood. It is shaped in a dual ring structure. Both openings are used when severing and reconnecting portions of DNA. The inner rings are lined similarly with bases to attract the negatively charged phosphate linkages of the DNA strands (Kysela 1). It also takes a highly spiraled shape due to secondary linkages between every third amino acid. However, the interactions which yield its complicated two gate form are still not fully known (Harkins 1).
There are multiple subgroups of DNA Topoisomerase II that act as cleaving and rejoining agents for multiple DNA strands (Bjornsti 1). The ways in which this process is enacted are various depending on the species and subtype of Topoisomerase II, but a general example can be given to illustrate the interactions. Similarly to Topoisomerase I, a DNA strand is summoned through the larger of Topoisomerase II's two holes. Two Tyrosine residues lining either side of the gate snip both ends of the phosphate backbone, severing the DNA strand completely (Goodsell, 2). Another DNA molecule is threaded through the second hole. The lack of tyrosine bonds on this end of the enzyme allows it to pass freely. As it passes, severs the covalent bonds holding the original cleaved DNA molecule to the tyrosine bonds. The two strands reconnect, and the resultant strand is a newly concatenated strand of DNA. Topoisomerase II is also capable of rethreading a looped DNA strand so that its coiled form can be untwisted into a single loop (Brutlag 9). It severs the strand and allows the stored energy to bring the nicked end from the back of the coil to the front. Once done, the other end of the severed coil passes through and rejoins to form the original loop. The movement from back to front changes the structure of the DNA loop, however, and solves the inherant topological problem posed by allowing the DNA to reshape itself into a simple loop, which can then be accessed more easily for reading or copying of the genetic information.
DNA Topoisomerase II serves a vital roll during DNA replication. When DNA is replicated in mitosis, it must be severed into two new strands by a 'fork', which works along the strand of DNA until it is halved. The difficulty with this method is that a supercoiled strand will become increasingly supercoiled as the fork moves, eventually bunching it up into an impossibly dense structure and halting the replication process (Wang 4). This difficulty is solved by positioning a DNA Topoisomerase II behind the forking replication. The fork rotates on its access to prevent tension from building on the forward end. The Topoisomerase II takes the two severed strands' positive supercoils and eliminates them as they pass through the enzyme's mechanism. Topoisomerase I cannot preform the same function, because it is not as efficient at untangling positive supercoils as it is with negative supercoils. These predictions have not yet been conclusively proven, but several studies show distinct evidence that Topoisomerase II is the dominant variety involved in the replication process (Wang 5).
Recent developments in DNA Topoisomerase have concentrated on applications to cancer treatment. The vital roll that they serve in DNA replication and thus cellular mitosis serves as an excellent weak point with which to attack. To combat tumors, drugs have been turning to topoisomerase inhibitors. They come in various forms: antibacterial, antiparasitic, antifungal, antiviral, but all take similar actions against topoisomerase. By stabilizing the cleaved state and rendering the covalent bond between the tyrosine residues and phosphate linkages of DNA strands permanent instead of temporary, inhibitors can prevent continued action on the part of the topoisomerase II. The exact chemical reactions which allow this stabilization are unknown, but it is theorized that the drugs bind with the DNA strands themselves to aid the stablization of the molecules until the concatenation process has been fully disrupted. Subsequently interference with DNA reproduction results in chromosomal loss and eventual cell death (Potmesil 15-16).
Topoisomerase I and II are essential to the function of all lifeforms on earth. They are present in both prokaryotic and eukaryotic cells and feature significantly in the reading, copying, and concatenation of genetic information. Topoisomerase I plays a heavy roll in relieving supercoiled tensions and easing the difficulty of transcribing genetic information. Topoisomerase II aids in concatenation and 'cleans up' after genetic replication by easing positive supercoils. Both enzymes function by the usage of basic residues along their inner surfaces to attract negatively charged portions of DNA backbones and sever linkages using the OH group of a tyrosine residue. There are two tyrosine residues in Topoisomerase II, allowing the cleaving of both backbones, and a single residue in Topoisomerase I, functioning to allow the swivel of DNA along its single connected phosphate linkage. Inhibiting topoisomerase function plays a useful role in cancer treatment. Through its unique structural properties, the topoisomerase enzymes help to continue the process of life with their genetic interactions and modifications.
Works Cited
Andoh,Toshiwo, Ikeda,Hideo, Oguro,Masao, ed., Molecular Biology of DNA Topoisomerases and Its Application to Chemotherapy. (Tokyo: CRC Press, 1991).
Kysela,David and Marcey,David. Topoisomerase I. OMM Exhibits. Internet. . September, 2002.
Fox,Fred C. DNA Replication and Recombination. (New York: Alan R. Liss, Inc., 1986)
Brutlag, Doug. DNA Topoisomerases. Advanced Molecular Biology. (Standford: Standford Press, 2000)
Wang,James C. "Cellular Roles of DNA Topoisomerases: A Molecular Perspective". Nature Magazine. (Hampshire: Nature Publishing Group, 2002)
Goodsell,David S. The Molecular Perspective: DNA Topoisomerase.
Champoux,James J. DNA Topology and DNA Topoisomerases. Internet. < http://faculty.washington.edu/champoux/>. Date not given.
Bjornsti,Mary-Ann and Osheroff,Neil ed., DNA Topoisomerase Protocols: DNA Topology and Enzymes. (Totowa, New Jersey: Humana Press, 1999).
"Topoisomerases." Flybase. Internet. . August, 2002.
Potmesil,Milan and Kohn,Kurt W. Dna Topoisomerases in Cancer. (New York: Oxford University Press, 1991).
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