The Mechanism of Supercoiled DNA Recognition by Eukaryotic Type I Topoisomerases: II. A Comparison of the Enzyme Interaction with Specific and Nonspecific Oligonucleotides

Data on the interaction of DNA type I topoisomerases from the murine and human placenta cells with specific and nonspecific oligonucleotides of various structures and lengths are summarized. The relative contributions of various contacts between the enzymes and DNA that have previously been detected by X-ray analysis to the total affinity of the topoisomerases for DNA substrates are estimated. Factors that determine the differences in the enzyme interactions with specific and nonspecific single- and double-stranded DNAs are revealed. The results of the X-ray analysis of human DNA topoisomerase I are interpreted taking into account data on the comprehensive thermodynamic and kinetic analysis of the enzyme interaction with the specific and nonspecific DNAs.     

The mechanism of the supercoiled DNA recognition by eukaryotic type I topoisomerases. I. The enzyme interaction with nonspecific oligonucleotides

Interaction of the DNA type I topoisomerases from the murine and human placenta cells with nonspecific oligonucleotides was analyzed. The contributions of strong and week nonspecific electrostatic, van der Waals's, and hydrophobic interactions, and hydrogen bonding of the enzymes to the complex formation with the single- and double-stranded DNAs were determined. The factors that determine the top-priority recognition of the topologically stressed DNA were revealed. The results were interpreted in comparison with the X-ray analysis data for human DNA topoisomerase I.     

The Mechanism of the Supercoiled DNA Recognition by the Eukaryotic Type I Topoisomerases: I. The Enzyme Interaction with Nonspecific Oligonucleotides

Interaction of the DNA type I topoisomerases from the murine and human placenta cells with nonspecific oligonucleotides was analyzed. The contributions of strong and week nonspecific electrostatic, van der Waals's, and hydrophobic interactions, and hydrogen bonding of the enzymes to the complex formation with the single- and double-stranded DNAs were determined. The factors that determine the top-priority recognition of the topologically stressed DNA were revealed. The results were interpreted in comparison with the X-ray analysis data for human DNA topoisomerase I.     

Role of topoisomerases in cytotoxicity induced by DNA ligand Hoechst-33342 and UV-C in a glioma cell line

DNA ligand Hoechst-33342 significantly enhances UV induced cytotoxicity in human glioma cell lines (BMG-1 & U-87) with supra additive increase in cell death, cytogenetic damage, cell cycle delay, apoptosis and inhibition of PLDR. Cytotoxicity of Hoechst-33342 arises due to its interference in the breakage-rejoining reaction of DNA topoisomerases by stabilization of cleavable complexes. Since topoisomerases have also been implicated in the generation of potentially lethal DNA breaks by interaction with various types of DNA damage including UV induced DNA lesions, we investigated in present studies the role of functional topoisomerases in the synergistic cytotoxicity of Hoechst-33342 and UV in a human glioma cell line (BMG-1). Topoisomerase I activity analyzed by the plasmid relaxation assay, was significantly enhanced upon UV irradiation, implying a possible role of this enzyme in the processing of UV induced lesions. However, this increase in the activity was reduced by >50% in cells incubated with Hoechst-33342 for 1 hr prior to irradiation. Imunoflowcytometric analysis of the chromatin bound topoisomerases I and II levels (cleavable complex) using topoisomerases I and II anti-antibodies showed a good correlation between the induction of apoptosis by Hoechst-33342 and UV and enhancement in the level of topoisomerase II mediated cleavable complexes. Induction of apoptosis was associated with a decline in the level of Bcl2. Taken together, these studies show that supra additive cytotoxic effects of UV-C and Hoechst-33342 in BMG-1 cells are consequences of enhanced stabilization of topo II mediated cleavable complexes and alterations in specific signal transduction pathways of apoptosis, besides the inhibition of topoisomerase mediated repair processes.     

Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA.

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA. The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.     

Eukaryotic topoisomerases recognize nucleic acid topology by preferentially interacting with DNA crossovers.

Eukaryotic topoisomerases recognize DNA topology and preferentially react with positively or negatively supercoiled molecules over relaxed substrates. To elucidate the mechanism of this recognition, we examined the interaction of topoisomerases with DNA by electron microscopy. Under all conditions employed, approximately 90% of the bound type I or II enzyme was observed at points of helix--helix juxtaposition on negatively supercoiled plasmids which contained as few as four crossovers. Recognition was independent of torsional stress, as enzyme molecules were also found at crossovers on linear DNA. Since juxtaposed helices are more prevalent in supercoiled compared with relaxed nucleic acids, we propose that eukaryotic topoisomerases I and II recognize underwound or overwound substrates by interacting preferentially with DNA crossovers. This may represent a general mechanism for the recognition of DNA topology by proteins.     

Structural, thermodynamic, and kinetic basis for the activities of some nucleic acid repair enzymes

X-ray structural analysis provides no quantitative estimate of the relative contribution of specific and nonspecific or strong and weak interactions to the total affinity of enzymes for nucleic acids. We have shown that the interaction between enzymes and long nucleic acids at the molecular level can be successfully analyzed by the method of stepwise increase in ligand complexity (SILC). In the present review we summarize our studies of human uracil DNA glycosylase and apurinic/apyrimidinic endonuclease, E. coli 8-oxoguanine DNA glycosylase and RecA protein using the SILC approach. The relative contribution of structural (X-ray analysis data), thermodynamic, and catalytic factors to the discrimination of specific and nonspecific DNA by these enzymes at the stages of complex formation, the following changes in DNA and enzyme conformations and especially the catalysis of the reactions is discussed.     

An engineered mutant of vaccinia virus DNA topoisomerase I is sensitive to the anti-cancer drug camptothecin.

Although highly homologous to the other eukaryotic type I DNA topoisomerases, vaccinia virus DNA topoisomerase I is distinct in its resistance to the anti-cancer drug camptothecin. After comparison of available sequences of sensitive and resistant type I topoisomerases, the aspartic acid at position 221 of vaccinia virus topoisomerase I is mutated to a valine. The resulting mutant protein is partially active. In contrast to the wild type enzyme, the relaxation of supercoiled DNA is inhibited by camptothecin. Its cleavage reaction with DNA is enhanced by camptothecin due to inhibition of religation of DNA. This demonstrates that even though the size of vaccinia virus is only about one-third that of the other camptothecin-sensitive topoisomerases, it has a potential interaction site for camptothecin.     

Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro.

The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.     

Inhibition of Mycobacterium smegmatis topoisomerase I by specific oligonucleotides

DNA topoisomerase I from Mycobacterium smegmatis unlike many other type I topoisomerases is a site specific DNA binding protein. We have investigated the sequence specific DNA binding characteristics of the enzyme using specific oligonucleotides of varied length. DNA binding, oligonucleotide competition and covalent complex assays show that the substrate length requirement for interaction is much longer ( approximately 20 nucleotides) in contrast to short length substrates (eight nucleotides) reported for Escherichia coli topoisomerase I and III. P1 nuclease and KMnO(4) footprinting experiments indicate a large protected region spanning about 20 nucleotides upstream and 2-3 nucleotides downstream of the cleavage site. Binding characteristics indicate that the enzyme interacts efficiently with both single-stranded and double-stranded substrates containing strong topoisomerase I sites (STS), a unique property not shared by any other type I topoisomerase. The oligonucleotides containing STS effectively inhibit the M. smegmatis topoisomerase I DNA relaxation activity.     

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes.     

The Role of Weak Specific and Nonspecific Interactions in Enzymatic Recognition and Conversion of Long DNAs

According to the currently accepted model, enzymes searching for specific recognition sequences or structural elements (modified nucleotides, breaks, single-stranded DNA fragments, etc.) slide at a high rate along DNA. Such sliding is possible only if the enzymes possess sufficiently high affinity for all DNA, sequence notwithstanding. Therefore, significant differences in their affinity for specific and nonspecific DNA sequences are unlikely, and the formation of a complex between an enzyme and its target DNA is not a basic factor of enzyme specificity. To elucidate such factors, we have analyzed many DNA replication, DNA repair, topoisomerization, integration, and recombination enzymes using a number of physicochemical methods, including the method of stepwise increase in ligand complexity developed in our laboratory. It has been shown that high affinity of all studied enzymes for long DNAs is provided by the formation of many weak contacts of the enzyme with all nucleotide units covered by the protein globule. The main role lies in the contact between positively charged amino acid residues and internucleoside phosphate groups; however, the contribution of each contact is very small, and the full contact interface usually resembles that characteristic of interactions between oppositely charged biopolymer surfaces. In some cases, a significant contribution to the affinity is made through hydrophobic and/or van der Waals interactions of the enzymes with nucleotide bases. On the whole, such nonspecific interactions provide for five to eight orders of enzyme affinity for DNA, depending on the enzyme. Specific interactions of enzymes with long DNAs, in contrast to their contacts with small ligands, are usually weak and comparable in efficiency with weak nonspecific contacts. The sum of specific interactions most often provides for approximately one or, rarely, two orders of affinity. According to structural data, DNA binding to any of the investigated enzymes is followed by a stage of DNA conformation adjustment, which includes partial or complete DNA melting, deformation of its backbone, stretching, compression, bending or kinking, eversion of nucleotides from the DNA helix, etc. The full set of such changes is specific for each individual enzyme. The fact that all enzyme-dependent changes in DNA are effected through weak specific (rather than strong) interactions is very important. Enzyme-specific changes in DNA conformation are required for effective adjustment of reacting orbitals to an accuracy of 10°–15°, which is possible only in the case of specific DNAs. A transition from nonspecific to specific DNA leads to an increase in the reaction rate (k _cat) by four to eight orders of magnitude. Thus, the stages of DNA conformation adjustment and catalysis proper provide for the high specificity of enzyme action.     

Human adenovirus proteinase: DNA binding and stimulation of proteinase activity by DNA.

The interaction of the human adenovirus proteinase (AVP) with various DNAs was characterized. AVP requires two cofactors for maximal activity, the 11-amino acid residue peptide from the C-terminus of adenovirus precursor protein pVI (pVIc) and the viral DNA. DNA binding was monitored by changes in enzyme activity or by fluorescence anisotropy. The equilibrium dissociation constants for the binding of AVP and AVP-pVIc complexes to 12-mer double-stranded (ds) DNA were 63 and 2.9 nM, respectively. DNA binding was not sequence specific; the stoichiometry of binding was proportional to the length of the DNA. Three molecules of the AVP-pVIc complex bound to 18-mer dsDNA and six molecules to 36-mer dsDNA. When AVP-pVIc complexes bound to 12-mer dsDNA, two sodium ions were displaced from the DNA. A Delta of -4.6 kcal for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy results from nonspecific interactions between the AVP-pVIc complex and DNA. The cofactors altered the interaction of the enzyme with the fluorogenic substrate (Leu-Arg-Gly-Gly-NH)2-rhodamine. In the absence of any cofactor, the Km was 94.8 microM and the kcat was 0.002 s(-1). In the presence of adenovirus DNA, the Km decreased 10-fold and the kcat increased 11-fold. In the presence of pVIc, the Km decreased 10-fold and the kcat increased 118-fold. With both cofactors present, the kcat/Km ratio increased 34000-fold, compared to that with AVP alone. Binding to DNA was coincident with stimulation of proteinase activity by DNA. Although other proteinases have been shown to bind to DNA, stimulation of proteinase activity by DNA is unprecedented. A model is presented suggesting that AVP moves along the viral DNA looking for precursor protein cleavage sites much like RNA polymerase moves along DNA looking for a promoter.     

The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevisiae

The ATP-independent type I and the ATP-dependent type II DNA topoisomerase of the yeast Saccharomyces cerevisiae have been purified to near homogeneity, and the purification procedures are reported. Both purified topoisomerases are single subunit enzymes with monomer weights of Mr = 90,000 and 150,000 for the type I and type II enzyme, respectively. Sedimentation and gel filtration data suggest that the type I enzyme is monomeric and the type II enzyme is dimeric. Similar to other purified eukaryotic topoisomerases, the yeast type I enzyme does not require a divalent cation for activity, but is stimulated 10-20-fold in the presence of 7-10 mM Mg(II) or Ca(II). Mn(II) is about 25% as efficient as Mg(II) in this stimulation but Co(II) is inhibitory. The yeast type II topoisomerase has an absolute requirement for a divalent cation: Mg(II) is the most effective, whereas Mn(II), Ca(II), or Co(II) supports the reaction to a lesser extent. The type II enzyme also requires ATP or dATP; the nonhydrolyzable ATP analogues adenylyl imidodiphosphate and adenylyl (beta,gamma-methylene)diphosphonate are potent inhibitors. Both yeast topoisomerases are completely inhibited by N-ethylmaleimide at 0.5 mM. In addition, the type II enzyme, but not the type I enzyme, is inhibited to various extents by coumermycin, ethidium, and berenil. Both topoisomerases are nuclear enzymes; no topoisomerase specific to mitochondria has been detected.     

Mapping of class I DNA sequences within the human major histocompatibility complex

Mutants that had lost expression of alleles of one or more HLA loci were isolated with immunoselection after gamma-irradiation of a human lymphoblastoid cell line LCL 721. DNAs from the mutants were digested with restriction endonucleases and analyzed by Southern blotting using probes for class I HLA genes. Eight polymorphic cut sites for HindIII and PvuII were discovered in class I-associated sequences of LCL 721. Losses of specific fragments generated by restriction enzymes could be associated with losses of specific antigenic expressions and it was possible in this way to assign HLA-A1, HLA-A2, and HLA-B8 to specific DNA fragments. Patterns of gamma-ray-induced segregations of DNA fragments permitted rough linkage alignment of about 30% of the fragments generated by PvuII. The resultant map showed that there are class I HLA genes on the telomeric side of the HLA-A locus. Restriction enzyme site polymorphisms were also examined in a panel of DNAs isolated from peripheral blood lymphocytes (PBLs) of HLA-typed individuals. This panel of PBL DNA complemented the analysis using the HLA deletion mutants.     

DNA topoisomerase I cleavage sites in DNA and in nucleoprotein complexes.

The intracellular substrate for eukaryotic DNA topoisomerases is chromatin rather than protein-free DNA. Yet, little is known about the action of topoisomerases on chromatin-associated DNA. We have analyzed to what extent the organization of DNA in chromatin influences the accessibility of DNA molecules for topoisomerase I cleavage in vitro. Using potassium dodecyl sulfate precipitation (Trask et al., 1984), we found that DNA in chromatin is cleaved by the enzyme with somewhat reduced efficiency compared to protein-free DNA. Furthermore, using native SV40 chromatin and mononucleosomes assembled in vitro, we show that DNA bound to histone octamer complexes is cleaved by topoisomerase I and that the cleavage sites as well as their overall distribution are identical in histone-bound and in protein-free DNA molecules.     

Computer analysis of conformational and physicochemical percularities of sequences cleaved by DNA topoisomerase I

After complexation of DNA with enzymes a specific adaptation of DNA structure including its partial or nearly complet melting, change of sugar-phosphate backbone structure, stretching, compression, bending or kinking, flipping out of nucleotides from the DNA helix, etc. take place. The full set of such changes is specific for each individual enzyme and is a very important for effective adjustment of reacting orbitals of enzyme and specific DNA atoms with accuracy up to 10-15 degrees. Efficiency of DNA sequence adaptation in the direction providing by enzyme depends on many specific structural characteristics of DNA. Maximal adjustment of DNA structure can be achieved only for specific sequences, therefore on going from nonspecific to specific DNAs the increase of the catalytic rate by 4-8 orders of magnitude takes place. DNA topoisomerase I is a sequence-dependent enzyme, but it can cleave with lower efficiency DNA sequences, which are significantly different from an optimal one. We have carried out the computer analysis of structural characteristics of many DNA sequences utilizing by topoisomerase using the method which is based on the analysis of conformational and physico-chemical characteristics of DNA helix and gives a detailed information about similarities or differences of DNA structural units. In addition to such characteristics as base tilt angle, shift of base pair, helix steering angle, and helix step for all cleaved sequences the presence of sterically disadvantageous contacts in small grove between N3 and NH2 of guanines and N3 of adenines were detected which corresponds to the presence Py-Pu dinucleotides in the cleavaged site. In addition, for optimal sequences bending of DNA helix toward major groove is characterized. The proposed method seems to be a very perspective for the analysis of an efficiency of nucleic acids cleavage by different DNA- and RNA-dependent enzymes.     

DNA topoisomerase III from extremely thermophilic archaebacteria. ATP-independent type I topoisomerase from Desulfurococcus amylolyticus drives extensive unwinding of closed circular DNA at high temperature.

A second type I topoisomerase was purified from the extremely thermophilic archaebacterium Desulfurococcus amylolyticus. In contrast to the previously described reverse gyrase from this organism, the novel enzyme designated as Dam topoisomerase III is an ATP-independent relaxing topoisomerase. It is a monomer with Mr 108,000, as determined by electrophoresis under denaturing conditions and by size exclusion chromatography. Dam topoisomerase III, like other bacterial type I topoisomerases, absolutely requires Mg2+ for activity and is specific for single-stranded DNA. At 60-80 degrees C, it relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA. At 95 degrees C, the enzyme unwinds both positively and negatively supercoiled substrates and produces extensively unwound form I* and I** DNA. The peculiarities of DNA topoisomerization at high temperatures are discussed.     

Archaebacterial reverse gyrase cleavage-site specificity is similar to that of eubacterial DNA topoisomerases I.

ATP-dependent type I topoisomerases from extremely thermophilic archaebacteria--reverse gyrases--drive positive supercoiling of DNA. We showed that reverse gyrase from Desulfurococcus amylolyticus breaks the DNA at specific sites and covalently binds to the 5' end. In 30 out of 31 sites located in pBR322 DNA fragments, cleavage occurs at the sequence 5'---CNNN/---(N is any base). The same rule was previously shown to hold for single-stranded DNA breakage by eubacterial topoisomerases I. The relative cleavage frequencies at different sites depend on Mg2+ and temperature. We discuss the possible physiological and mechanistic role of the above specificity of the bacterial topoisomerases I.