Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro.

Purified vaccinia virus DNA topoisomerase I forms a cleavable complex with duplex DNA at a conserved sequence element 5'(C/T)CCTTdecreases in the incised DNA strand. DNase I footprint studies show that vaccinia topoisomerase protects the region around the site of covalent adduct formation from nuclease digestion. On the cleaved DNA strand, the protected region extends from +13 to -13 (+1 being the site of cleavage). On the noncleaved strand, the protected region extends from +13 to -9. Similar nuclease protection is observed for a mutant topoisomerase (containing a Tyr ---- Phe substitution at the active site amino acid 274) that is catalytically inert and does not form the covalent intermediate. Thus, vaccinia topoisomerase is a specific DNA binding protein independent of its competence in transesterification. By studying the cleavage of a series of 12-mer DNA duplexes in which the position of the CCCTTdecreases motif within the substrate is systematically phased, the "minimal" substrate for cleavage has been defined; cleavage requires six nucleotides upstream of the cleavage site and two nucleotides downstream of the site. An analysis of the cleavage of oligomer substrates mutated singly in the CCCTT sequence reveals a hierarchy of mutational effects based on position within the pentamer motif and the nature of the sequence alteration.     

Residues within the N-terminal domain of human topoisomerase I play a direct role in relaxation

All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme.     

Structure of a complex between E. coli DNA topoisomerase I and single-stranded DNA

In order to gain insights into the mechaism of ssDNA binding and recognition by Escherichia coli DNA topoisomerase I, the structure of the 67 kDa N-terminal fragment of topoisomerase I was solved in complex with ssDNA. The structure reveals a new conformational stage in the multistep catalytic cycle of type IA topoisomerases. In the structure, the ssDNA binding groove leading to the active site is occupied, but the active site is not fully formed. Large conformational changes are not seen; instead, a single helix parallel to the ssDNA binding groove shifts to clamp the ssDNA. The structure helps clarify the temporal sequence of conformational events, starting from an initial empty enzyme and proceeding to a ssDNA-occupied and catalytically competent active site.     

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.     

Recombinogenic flap ligation mediated by human topoisomerase I

Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3'-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks. Here, we characterize flap ligation mediated by human topoisomerase I. We demonstrate that cleavage complexes containing the enzyme at a blunt end allow invasion of a 3'-acceptor tail matching the scissile strand of the donor, which facilitates ligation of the recessed 5'-hydroxyl end. However, the reaction was strictly dependent on the length of double-stranded DNA of the donor complexes, and longer stretches of base-pairing inhibited strand invasion. The stabilization of the DNA helix was most probably provided by the covalently bound enzyme itself, since deleting the N-terminal domain of human topoisomerase I stimulated flap ligation. We suggest that stabilization of the DNA duplex upon enzyme binding may play an important role during normal topoisomerase I catalysis by preventing undesired strand transfer reactions. For flap ligation to function in a repair pathway, factors other than topoisomerase I, such as helicases, would be necessary to unwind the DNA duplex and allow strand invasion.     

Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I

Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently. Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA. These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria. Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature. Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation. Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission. Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies. The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein. Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions. This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme. Noncovalent binding is therefore independent of competence in transesterification. It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes.     

Crystal structure of a bacterial type IB DNA topoisomerase reveals a preassembled active site in the absence of DNA

Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-A crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) beta-sheet domain (amino acids 1-90) and a predominantly alpha-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an "open" conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.     

Identification of an N-terminal domain of eukaryotic DNA topoisomerase I dispensable for catalytic activity but essential for in vivo function.

We have found that deletion of a 70-amino acid domain, spanning from position 141 to 210 in the N-terminal part of human topoisomerase I, has no effect on the catalytic activity of the enzyme in vitro but suppresses the lethal consequence of overexpressing human topoisomerase I in a rad52 top1 Saccharomyces cerevisiae strain. By immunostaining, the 70-amino acid domain is shown to be necessary for nuclear location of topoisomerase I. We demonstrate that the nuclear localization signal from the SV40 large T antigen can substitute for the 70-amino acid domain, restoring both the lethal effect of overexpression and the correct subcellular localization of topoisomerase I. Thus, we have identified a domain in the N-terminal part of human topoisomerase I, nonessential for catalytic activity in vitro but serving an in vivo function by directing the enzyme to the nucleus. Based on sequence comparisons, we suggest that this domain is a conserved element in the apparently non-homologous N-terminal parts of yeast and human topoisomerase I.     

Ability of viral topoisomerase II to discern the handedness of supercoiled DNA: bimodal recognition of DNA geometry by type II enzymes

Previous studies with human and bacterial topoisomerases suggest that the type II enzyme utilizes two distinct mechanisms to recognize the handedness of DNA supercoils. It has been proposed that the ability of some type II enzymes, such as human topoisomerase IIalpha and Escherichia coli topoisomerase IV, to distinguish supercoil geometry during DNA relaxation is mediated by elements in the variable C-terminal domain of the protein. In contrast, the ability of human topoisomerase IIalpha and topoisomerase IIbeta to discern the handedness of supercoils during DNA cleavage suggests that residues in the conserved N-terminal or central domain of the protein are involved in this process. To test this hypothesis, the ability of Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) topoisomerase II to relax and cleave negatively and positively supercoiled plasmids was assessed. These enzymes display a high degree of sequence identity with the N-terminal and central domains of eukaryotic topoisomerase II but naturally lack the C-terminal domain. While PBCV-1 and CVM-1 topoisomerase II relaxed under- and overwound substrates at similar rates, they were able to discern the handedness of supercoils during the cleavage reaction and preferentially cut negatively supercoiled DNA. Preferential cleavage was not due to a change in site specificity, DNA binding, or religation. These findings are consistent with a bimodal recognition of DNA geometry in which topoisomerase II uses elements in the C-terminal domain to sense the handedness of supercoils during DNA relaxation and elements in the conserved N-terminal or central domain during DNA cleavage.     

Site-specific DNA cleavage by vaccinia virus DNA topoisomerase I. Role of nucleotide sequence and DNA secondary structure.

Cleavage of linear duplex DNA by purified vaccinia virus DNA topoisomerase I occurs at a conserved sequence element (5'-C/T)CCTT decreases) in the incised DNA strand. Oligonucleotides spanning the high affinity cleavage site CCCTT at nucleotide 2457 in pUC19 DNA are cleaved efficiently in vitro, but only when hybridized to a complementary DNA molecule. As few as 6 nucleotides proximal to the cleavage site and 6 nucleotides downstream of the site are sufficient to support exclusive cleavage at the high affinity site (position +1). Single nucleotide substitutions within the consensus pentamer have deleterious effects on the equilibria of the topoisomerase binding and DNA cleavage reactions. The effects of base mismatch within the pentamer are more dramatic than are the effects of mutations that preserve base complementarity. Competition experiments indicate that topoisomerase binds preferentially to DNA sites containing the wild-type pentamer element. Single-stranded DNA containing the sequence CCCTT in the cleaved stand is a more effective competitor than is single-stranded DNA containing the complementary sequence in the noncleaved strand.     

Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase I.

The ability of a eukaryotic DNA topoisomerase I to catalyze DNA rearrangements was examined in vitro using defined substrates and purified enzyme. Site-specific DNA strand cleavage by vaccinia topoisomerase I across from a nick generated double-strand breaks that could be religated to a heterologous blunt-ended duplex DNA regardless of the sequence of the acceptor molecule. Topoisomerase bound covalently at internal positions could religate the bound strand to an incoming acceptor provided that DNA molecule had sequence homology to the region 3' of the scissile bond. These end-joining reactions suggest two potential modes of topoisomerase-mediated recombination that differ in their requirements for DNA homology.     

Rapid and prolonged stalling of human DNA topoisomerase I in UVA-irradiated genomic areas

DNA topoisomerase I appears to be involved in DNA damage and repair in a complex manner. The enzyme is required for DNA maintenance and repair, but it may also damage DNA through its covalently DNA-bound, catalytic intermediate. The latter mechanism plays a role in tumor cell killing by camptothecins, but seems also involved in oxidative cell killing and certain stages of apoptosis. Stalling and/or suicidal DNA cleavage of topoisomerase I adjacent to nicks and modified DNA bases has been demonstrated in vitro. Here, we investigate the enzyme's interactions with UVA-induced DNA lesions inside living cells. We irradiated cells expressing GFP-tagged topoisomerase I with an UVA laser focused through a confocal microscope at confined areas of the nuclei. At irradiated sites, topoisomerase I accumulated within seconds, and accumulation lasted for more than 90 min. This effect was apparently due to reduced mobility, although the enzyme was not immobilized at the irradiated nuclear sites. Similar observations were made with mutant versions of topoisomerase I lacking the active site tyrosine or the N-terminal domain, but not with the N-terminal domain alone. Thus, accumulation of topoisomerase I at UVA-modified DNA sites is most likely due to non-covalent binding to damaged DNA, and not suicidal cleavage of such lesions. The rapid onset of accumulation suggests that topoisomerase I functions in this context as a component of DNA damage recognition and/or a cofactor of fast DNA-repair processes. However, the prolonged duration of accumulation suggests that it is also involved in more long-termed processes.     

Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.     

Single-strand DNA cleavages by eukaryotic topoisomerase II

A new purification method for eukaryotic type II DNA topoisomerase (EC 5.99.1.3) is described, and the avian enzyme has been purified and characterized. An analysis of the cleavage reaction has revealed that topoisomerase II can be trapped as a DNA-enzyme covalent complex containing DNA with double-stranded and single-stranded breaks. The data indicate that DNA cleavage by topoisomerase II proceeds by two asymmetric single-stranded cleavage and resealing steps on opposite strands (separated by 4 bp) with independent probabilities of being trapped upon addition of a protein denaturant. Single-strand cleavages were directly demonstrated at both strong and weak topoisomerase II sites. Thus, a match to the vertebrate topoisomerase II consensus sequence (sequence; see text) (N is any base, and cleavage occurs between -1 and +1) [Spitzner, J.R., & Muller, M.T. (1988) Nucleic Acids Res. 16, 5533-5556)] does not predict whether a cleavage site will be single stranded or double stranded; however, sites cleaved by topoisomerase II that contain two conserved consensus bases (G residue at +2 and T at +4) generally yield double-strand cleavage whereas recognition sites lacking these two consensus elements yield single-strand cleavages. Finally, single-strand cleavages with topoisomerase II do not appear to be an artifact caused by damaged enzyme molecules since topoisomerase II in freshly prepared, crude extracts also shows the property of single-strand cleavages.     

DNA topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus with specific DNA cleavage activity

We report the production, purification, and characterization of a type IA DNA topoisomerase, previously designated topoisomerase I, from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was capable of relaxing negatively supercoiled DNA at 75 degrees C in the presence of Mg2+. Mutation of the putative active site Tyr318 to Phe318 led to the inactivation of the protein. The S. solfataricus enzyme cleaved oligonucleotides in a sequence-specific fashion. The cleavage occurred only in the presence of a divalent cation, preferably Mg2+. The cofactor requirement of the enzyme was partially satisfied by Cu2+, Co2+, Mn2+, Ca2+, or Ni2+. It appears that the enzyme is active with a broader spectrum of metal cofactors in DNA cleavage than in DNA relaxation (Mg2+ and Ca2+). The enzyme-catalyzed oligonucleotide cleavage required at least 7 bases upstream and 2 bases downstream of the cleavage site. Analysis of cleavage by the S. solfataricus enzyme on a set of oligonucleotides revealed a consensus cleavage sequence of the enzyme: 5'-G(A/T)CA(T)AG(T)G(A)X / XX-3'. This sequence bears more resemblance to the preferred cleavage sites of topoisomerases III than to those of topoisomerases I. Based on these data and sequence analysis, we designate the enzyme S. solfataricus topoisomerase III.     

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.     

The N-terminus of m5C-DNA methyltransferase MspI is involved in its topoisomerase activity.

DNA cytosine methyltransferase MspI (M.MspI) must require a different type of interaction of protein with DNA from other bacterial DNA cytosine methyltransferases (m5C-MTases) to evoke the topoisomerase activity that it possesses in addition to DNA-methylation ability. This may require a different structural organization in the solution phase from the reported consensus structural arrangement for m5C-MTases. Limited proteolysis of M.MspI, however, generates two peptide fragments, a large one (p26) and a small one (p18), consistent with reported m5C-MTase structures. Examination of the amino-acid sequence of M.MspI revealed similarity to human topoisomerase I at the N-terminus. Alignment of the amino-acid sequence of M.MspI also uncovered similarity (residues 245-287) to the active site of human DNA ligase I. To evaluate the role of the N-terminus of M.MspI, 2-hydroxy-5-nitrobenzyl bromide (HNBB) was used to truncate M.MspI between residues 34 and 35. The purified HNBB-truncated protein has a molecular mass of approximately equal 45 kDa, retains DNA binding and methyltransferase activity, but does not possess topoisomerase activity. These findings were substantiated using a purified recombinant MspI protein with the N-terminal 34 amino acids deleted. Changing the N-terminal residues Trp34 and Tyr74 to alanine results in abolition of the topoisomerase I activity while the methyltransferase activity remains intact.     

Major groove interactions of vaccinia Topo I provide specificity by optimally positioning the covalent phosphotyrosine linkage

Vaccinia DNA topoisomerase (vTopo) is a prototypic eukaryotic type I topoisomerase that shows high specificity for nucleophilic substitution at a single phosphodiester linkage in the pentapyrimidine recognition sequence 5'-(C/T)+5 C+4 C+3 T+2 T+1 p / N(-1). This reaction involves reversible transesterification where the active site tyrosine of the enzyme and a 5'-hydroxyl nucleophile of DNA compete for attack at the phosphoryl group. The finite lifetime of the covalent phosphotyrosine adduct allows the enzyme to relax multiple supercoils by rotation of the 5'-OH strand before the DNA backbone is religated. To dissect the nature of the unique sequence specificity, subtle modifications to the major groove of the GGGAA 5'-sequence of the nonscissile strand were introduced and their effects on each step of the catalytic cycle were measured. Although these modifications had no effect on noncovalent DNA binding (K(D)) or the rate of reversible DNA cleavage (k(cl)), significant decreases in the cleavage equilibrium (K(cl) = k(cl)/k(r)) arising from increased rates of 5'-hydroxyl attack (k(r)) at the phosphotyrosine linkage were observed. These data and other findings support a model in which major groove interactions are used to position the phosphotyrosine linkage relative to the mobile 5'-hydroxyl nucleophile. In the absence of native sequence interactions, the phosphotyrosine has a higher probability of encountering the 5'-hydroxyl nucleophile, leading to an enhanced rate of ligation and a diminished equilibrium constant for cleavage. By this unusual specificity mechanism, the enzyme prevents formation of stable covalent adducts at nonconsensus sites in genomic DNA.