Chinese name: topoisomerase mbth: Isomerase Introduction: The characteristics of a class of enzymes existing in the nucleus: they can catalyze the break and combination of DNA chains. Brief introduction, clinical use, classification, category I, category II, chemical reaction, use, function, new discovery, brief introduction DNA topoisomerase is a kind of enzyme existing in the nucleus, which can catalyze the breakage and combination of DNA chains, thus controlling the topological state of DNA. Topoisomerase participates in supramolecules. There are mainly two kinds of topoisomerases in mammals. DNA topoisomerase I catalyzes the change of topological isomeric state of DNA replication by forming a short single-strand cleavage-binding cycle; On the contrary, topoisomerase II can change the topological state of DNA by causing the instantaneous double-stranded enzyme bridge to break, then opening and closing again. Mammalian topoisomerase Ⅱ can be divided into α Ⅱ type and β Ⅱ type. The antitumor activity of topoisomerase toxin is related to its stability to enzyme -DNA cleavable complex. These drugs can effectively convert enzymes into fibrinolysis by stabilizing the enzyme -DNA cleavable complex. These drugs used in clinic include adriamycin, actinomycin D, daunorubicin, VP- 16, VM-26 (teniposide or epipodophyllotoxin). Relatively speaking, bis-benzimidazole and tetrabenzimidazole are more commonly used as mammalian isomerase type II toxins (Chen et al., Cancer Res.1993,53,1332-1335; Sun et al, Journal of Medical Chemistry 1995, 38, 3638-3644; Jin et al., doctor of medicine. Chemistry. 1996,39,992-998), some chelidonine alkaloids (benzo [c] benzoanthracopyridine) and protoberberine alkaloids and their synthetic analogues (Makhey et al., Med. Chemistry. Resolution 65438+). Janin et al., J.Med.Chem. 1975,18,708-713; Makhey et al., Bioorg. & Med.chem. 1996, 4, 78 1-79 1), and bulgerain(Fujii et al., J.Biol.Chem. 1993, 268,) and sainin(Yamashita 5838-5845) and indolocarbazole (Yamashita et al., Biochemistry1992,31,12069- 12075). Other topoisomerase toxins that have been identified include certain alkaloids of chelidonine and cinnoline compounds (see LaVoie et al., U.S. Pat. No.6 140328 and WO 001/32631). Although these compounds have great uses, their application is limited because of their low solubility. At the end of June 5438+ 10, 2006, the U.S. Patent Office awarded four patents to the University of New Jersey, which were the synthesis and application of amino and nitro substituted drugs for isomerase. Classification can be divided into two categories: one is called topoisomerase I, and the other is called topoisomerase II. Topoisomerase I catalyzes the breakage and reconnection of DNA strands, and only acts on one strand at a time, that is, it catalyzes the instantaneous breakage and reconnection of single strand. They do not need energy cofactors, such as ATP or NAD. Escherichia coli DNA topoisomerase I is also called ω protein, and mouse liver DNA topoisomerase I is also called incision closing enzyme. Topoisomerase II can simultaneously break and connect two DNA strands. They usually need the energy cofactor ATP. In topoisomerase ⅱ, it can be divided into two subclasses: one subclass is DNA gyrase, whose main function is to introduce negative supercoils and play a very important role in DNA replication. So far, DNA gyrase has only been found in prokaryotes. Another subclass is to transform supercoiled DNA (including positive supercoiled DNA and negative supercoiled DNA) into a relaxed form without supercoiled DNA. Although this reaction is a thermodynamically favorable direction, I don't know why they still need ATP like DNA gyrase, which may be related to restoring the conformation of the enzyme. This enzyme exists in both prokaryotes and eukaryotes. The first kind of DNA topoisomerase can catalyze many reactions, which can only be briefly described here. The affinity of DNA topoisomerase I for single-stranded DNA is much higher than that for double-stranded, which is the molecular basis for its recognition of negative supercoiled DNA, because negative supercoiled DNA often has a certain degree of single-stranded region. The higher the negative supercoil, the faster the DNA topoisomerase I acts. It is now known that the negative supercoil in organisms is stable at around 5%, and it will not work if it is low or high. The organism makes the negative supercoil reach a stable state through the opposite action of topoisomerase 1 and II. It has been found that the mutation of gene A encoding E.coli topoisomerase I will cause compensatory mutation of gyrase gene. Otherwise, negative supercoils increase and cell viability decreases. The base sequence specificity of topoisomerase I is not high, but the tangent point must be 4 bases downstream of C (including C itself). After the single strand of DNA is cut off, topoisomerase I attaches to the 5- end of the incision, and the energy of hydrolyzing phosphodiester bond is stored for attaching the incision, so the function of topoisomerase I does not need energy supply. In addition, topoisomerase I can also promote the renaturation of two single strands, and its function is to reduce the negative pressure of the number of strands produced in the renaturation process and make the renaturation process go through to the end. If one part of a single link is cut off and the other part bypasses the incision, a trefoil structure molecule can be produced. If there are two double strands, and one of them has a gap, topoisomerase 1 can cut the chain opposite to the gap, put the complete double strands in, and then connect them to form a chain. The above three reactions are shown on the right. Topoisomerase I can also catalyze other reactions, which will be discussed in the mechanism of replication and recombination. The topoisomerase II (gyrase) of the second Escherichia coli has the ability to form or decompose double-stranded DNA loops and binding molecules in addition to introducing negative supercoils. Class ⅱ topoisomerases have no base sequence specificity, and they can bind any two pairs of crossed double-stranded DNA. DNA gyrase has two α subunits and two β subunits. The α subunit is about 105KDa, which is encoded by gyrA gene and has phosphodiesterase activity, which can be inhibited by nalidixic acid. Subunit A is about 95KD, encoded by graB gene, which has ATPase activity and can be inhibited by neomycin. Both drugs can inhibit DNA replication of wild-type Escherichia coli. It can be seen that DNA gyrase is indispensable for E.coli to replicate after cutting DNA double strands. Two A subunits are bound at the 5' end of the incision, and the energy obtained by hydrolyzing phosphodiester bonds is stored. Because of the integrity of the enzyme, it is impossible for the four ends of the DNA chain to rotate at will. Due to the allosteric effect of the enzyme, the intact double strand passes through the incision and then forms the phosphodiester bond again. The function of β subunit is to hydrolyze ATP and restore the original conformation of enzyme molecules for the next round of reaction. This can be confirmed by replacing ATP with ATP homologue β, γ-imino ATP. Because this homologue can not be hydrolyzed by DNA gyrase, it can promote the first round of topological isomerization reaction, increase negative supercoils and hinder further topological isomerization reaction in the future. Chemical reaction DNA topoisomerase catalyzes many reactions, the essence of which is to cut off the phosphodiester bond of DNA first, then change the number of DNA connections before connecting, and it has the functions of endonuclease and DNA ligase. However, they cannot connect pre-existing broken DNA, that is, their cleavage reaction and connection reaction are coupled with each other. Topoisomerase (including type I and type II) can be explained by the sign conversion model (bottom left). In addition to DNA topoisomerase, many reagents, especially platelet dye molecules, can be embedded between adjacent bases, affecting base aggregation and changing the topological state of DNA. The most obvious example is ethidium bromide. For example, taking the binding test of CCC molecule of SV40 with ethidium bromide as an example, in the absence of dye, this DNA is negative supercoiled and its sedimentation constant is very high (21s); When the ratio of dye molecules to nucleotides is 0.05, the sedimentation number drops to l6S, and DNA is in a relaxed state without supercoils. When the ratio of dye molecules to nucleotides increases to 0.09, the sedimentation constant rises to about 2 1S, and DNA molecules are positive supercoils. This relationship is shown in the figure on the right, but it should be noted that ethidium bromide does not change the Lk value, but the embedding of ethidium bromide molecules increases the tight binding state of local DNA secondary structure. Therefore, with the increase of the number of embedded dye molecules, the negative supercoils first decrease and disappear, and then the positive supercoils increase. This is similar to the situation that single-stranded DNA binding protein promotes the transformation of negative supercoils into bubble structures. Isomerase: an enzyme that changes the sequence number of DNA by cutting the phosphodiester bond in one or two DNA chains, then rewinding and sealing it. DNA gyrase DNA isomerase is a general term for enzymes that catalyze the transformation of DNA topoisomers. In order to analyze the reaction mechanism in vitro, cyclic DNA was used as substrate to catalyze the coupling reaction of DNA chain disconnection and combination. In the topological transformation of closed-loop double-stranded DNA, one or two strands of DNA should be temporarily cut off, which can be divided into two types according to isomerization methods. The topoisomerase Ⅱ i that changes the topological structure by cutting one chain is called-isomerase Ⅱ i, and the topoisomerase Ⅱ that is carried out by cutting two chains is called. As a type I topoisomerase, there are ω-protein of Escherichia coli (composed of a single polypeptide chain with molecular weight of 1 10000) and notch closure enzyme (molecular weight of about 65000-70000, molecular weight of about 10000) existing in various eukaryotic cells. Type ⅱ topoisomerase includes DNA gyrase in bacteria, topoisomerase ⅱ in phage T4 and ATP-dependent topoisomerase ⅱ in eukaryotic cells. In addition, the irt gene product of λ phage and the gene A product of φX 174 phage also have the activity of cleaving binding enzyme, which can be considered as one of topoisomerases. Type I topoisomerase catalyzes isomerization without ATP energy. As an intermediate product of the reaction, in prokaryotes, the free 5'-OH terminal is connected with the 3'- phosphate terminal to form a valence bond with the enzyme, while in eukaryotes, the 5'- phosphate terminal at the 3'-OH terminal forms a valence bond with the enzyme. The energy stored in ester bonds may play a role in the recombination of broken ends. The reactions catalyzed by type I topoisomerase are as follows: In each cleavage and binding reaction, the L number of supercoiled DNA (see topoisomers of DNA) changes, that is, it relaxes. Complementary single-stranded circular DNA is transformed into double-stranded circular DNA with spiral structure, so that single-stranded DNA can be logically knotted or untied. In addition, when one strand of one molecule of two circular double-stranded DNA is cut, a chain-like cyclic dimer molecule (ca-tenane) is formed. In type ⅱ topoisomerase, DNA gyrase can catalyze closed-loop DNA to produce supercoils alone, which is unique. The other two enzymes can not only relax supercoils but also need ATP energy, and can also catalyze the catalytic reaction of gyrase. Eukaryotic topoisomerase ⅰ is involved in the formation of nucleosome, and bacterial ω protein is involved in transcription and the insertion of some transposons. Cyclooxygenase and T4 topoisomerase Ⅱ are involved in DNA replication and transcription. I (DNA topoisomerase I) catalyzes four reactions: ① relaxation of supercoils; (2) the formation of knots; ③ Formation of cyclic double-stranded molecules; ④ Connection of cyclic double-stranded molecules. This enzyme is derived from calf thymus, which is different from the enzyme derived from prokaryotes and shows activity even without Mg2+. Moreover, DNA topoisomerase I from prokaryotes only acts on negative-strand supercoiled molecules, and this enzyme can make both positive and negative supercoiled molecules form loose forms. Objective DNA type ⅱ topoisomerase ⅱ topoisomerase skillfully completed the process of opening the DNA double helix. It cuts the double helix structure of DNA and allows another helix to pass through the gap, and then a double helix is opened. The picture shown here is constructed from two kinds of protein: protein with the number 1bgw has the lower half structure of topoisomerase, and protein with the number 1eil comes from the domain of a gyrase, which is very similar to the upper half of topoisomerase. Topoisomerase has high catalytic activity, and it has some "gate" structures that control DNA to enter two cracks on it. Here, the two tyrosine shown in red combine with the DNA chain to form a valence bond, and this tight combination mode continues until the DNA is restored. The function is to relax the super spiral. The so-called supercoil is a form of tension accumulation in DNA. Topoisomerase inhibitor is an important anti-tumor drug, which is thought to play its role by stabilizing the valence complex formed between topoisomerase and DNA, and then set obstacles for DNA replication mechanism. Scientists still don't know much about the origin of drugs targeting topoisomerase. The cover picture of this issue shows the accumulation of positive DNA supercoils caused by this drug. This super entanglement of DNA will hinder the progress of a DNA polymerase, and may also prevent or destroy replication fork, leading to cell death. A new discovery of the interaction between DNA helix and topoisomerase An international research team led by the Dutch has cracked the mechanism of naturally releasing the twisted tension accumulated in DNA at the molecular level. Researchers from Delft University of Technology, French Teachers' College and Si Long-Kettering Institute published their research results in the March 3rd issue of Nature, which became the cover of this issue. Type IB topoisomerase can release the torque accumulated in DNA chain. During the research, researchers can track the activity of a single topoisomerase molecule on a single DNA molecule for a period of time at the molecular level. Topoisomerase can contain DNA, cut one of the two DNA strands, and stretch the DNA before reconnecting the sticky ends. With the help of sensitive detection instruments, researchers can measure different parameters, such as the friction of rotating DNA in the enzyme cavity. This study has a new understanding of the interaction between DNA and this enzyme. Two single strands of DNA are twisted together to form a double helix structure, while the base pair sequence of double strands stores genetic information. In the process of cell division, genetic material is copied, and the enzyme responsible for copying must be able to transcribe these base sequences. But to realize this process, the part of DNA that needs to be transcribed must be straightened. This winding and stretching of DNA molecules produces torsion, and the extent of torsion increases with the development of cell division. This force can delay the process of cell division and even stop it in some cases, and IB topoisomerase can reduce these torsional forces. The steps of this enzyme to release DNA twist are as follows: topoisomerase first clamps double-stranded DNA like a clamp, and then instantly passes through one of the two DNA chains. This distortion accumulated in DNA molecules then gradually disappears near the whole chain. After rotation, the enzyme grabs the rotated DNA again and reconnects the broken chain. Researchers were able to determine the exact number of supercoils separated by this topoisomerase between shearing and adhesion. The precise mechanism of IB topoisomerase is also of great significance to cancer research. Drugs that can inhibit the function of IB topoisomerase have been used in clinic, but their use may be improved after these findings are obtained.