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1.
Mycobacterium smegmatis topoisomerase I (MstopoI) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage in the absence of Mg2+, but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+ in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike MstopoI and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.  相似文献   

2.
Vaccinia virus DNA topoisomerase catalyzes reso­lution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5′-CCCTT↓, that are opposed within a partially mobile four-way junction. Efficient resolution occurs on a junction with a 10 bp segment of branch mobility (5′-GCCCTTATCG) that extends 4 bp 3′ of the scissile phosphate. Here we report that resolution is decreased when branch mobility is limited to an 8 bp segment extending 2 bp 3′ of the cleavage site and then eliminated when branch mobility is confined to the 6 bp GCCCTT sequence 5′ of the scissile phosphate. We surmise that a spacer region 3′ of CCCTT is needed for simultaneous cleavage at two opposing sites at the junction. Branch mobility is not required for reaction chem­i­stry at a junction, because topoisomerase cleaves a single CCCTT site in a non-mobile four-way junction where the scissile phosphate is at the crossover point. The junction resolvase activity of topo­isomerase may be involved in forming the hairpin telomeres of the vaccinia genome.  相似文献   

3.
Clerocidin (CL), a diterpenoid natural product, alkylates DNA through its epoxide moiety and exhibits both anticancer and antibacterial activities. We have examined CL action in the presence of topoisomerase IV from Streptococcus pneumoniae. CL promoted irreversible enzyme-mediated DNA cleavage leading to single- and double-stranded DNA breaks at specific sites. Reaction required the diterpenoid function: no cleavage was seen using a naphthalene-substituted analogue. Moreover, drug-induced DNA breakage was not observed using a mutant topoisomerase IV (ParC Y118F) unable to form a cleavage complex with DNA. Sequence analysis of 102 single-stranded DNA breaks and 79 double-stranded breaks revealed an overwhelming preference for G at the −1 position, i.e. immediately 5′ of the enzyme DNA scission site. This specificity contrasts with that of topoisomerase IV cleavage with antibacterial quinolones. Indeed, CL stimulated DNA breakage by a quinolone-resistant topoisomerase IV (ParC S79F). Overall, the results indicate that topoisomerase IV facilitates selective irreversible CL attack at guanine and that its cleavage complex differs markedly from that of mammalian topoisomerase II which promotes both irreversible and reversible CL attack at guanine and cytosine, respectively. The unique ability to form exclusively irreversible DNA breaks suggests topoisomerase IV may be a key intracellular target of CL in bacteria.  相似文献   

4.
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.  相似文献   

5.
The MazEF systems are thought to contribute to the capacity for long-term dormancy observed in the human pathogen, Mycobacterium tuberculosis. However, except for their functions as mRNA interferases, little is known regarding any additional cellular functions of these systems in the pathogen. In the present study, we observed a negative interplay between MazF protein Rv1495 and the sole M. tuberculosis DNA topoisomerase I (MtbTopA) with respect to protein functions. Through its C-terminal domain, MtbTopA physically interacted with and inhibited the mRNA cleavage activity of Rv1495. Rv1495, in turn, inhibited the DNA cleavage activity of MtbTopA as well as its function of relaxation of supercoiled DNA. An N-terminus fragment of Rv1495, designated Rv1495-N(29-56), lost mRNA cleavage activity, but retained a significant physical interaction and inhibitory effect on TopA proteins from both M. tuberculosis and M. smegmatis. This fragment, although less effective than the full-length protein, was able to inhibit mycobacterial growth when expressed through a recombinant plasmid in M. smegmatis. The Rv1495 physically interacted with the M. smegmatis TopA both in vitro and in vivo. Our findings imply that MazEF systems can affect bacterial survival by a novel mechanism that allows direct modulation of M. tuberculosis topoisomerase I.  相似文献   

6.
Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.  相似文献   

7.
The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme.  相似文献   

8.
Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I   总被引:12,自引:0,他引:12  
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.  相似文献   

9.
Several recent studies have shown that human topoisomerase I (htopoI) can recognize various DNA lesions and thereby form a covalent topoisomerase I–DNA complex, which is known to be detrimental to cells. We have investigated whether htopoI recognizes another htopoI that is covalently trapped on a DNA substrate. For this purpose we created an artificial DNA substrate containing a specific topoisomerase I binding sequence, where the enzyme was trapped in the covalently bound form. We demonstrate that, in vitro, free htopoI stimulates the formation of an additional cleavage complex immediately upstream of the covalently bound topoisomerase I. The predominant distance between the two cleavage sites is 13 nt. In addition we find that these two enzymes may form direct protein–protein contacts and we propose that these may be mediated through the formation of a dimer by domain swapping involving the C-terminal and the core domains. Finally, we discuss the possibility that the double cleavage reaction may be the initial step for the removal of the recognized cleavage complex.  相似文献   

10.
Antibacterial fluoroquinolones trap a cleavage complex of gyrase and topoisomerase (topo) IV inducing site-specific DNA breakage within a bent DNA gate engaged in DNA transport. Despite its importance for drug action and in revealing potential sites of topoisomerase catalysis, the mechanism of DNA selectivity is poorly understood. To explore its functional basis, we generated mutant versions of the strongly cleaved E-site and used a novel competitive assay to examine their gemifloxacin-mediated DNA breakage by Streptococcus pneumoniae topo IV and gyrase. Parallel studies of Ca2+-induced cleavage distinguished ‘intrinsic recognition’ of DNA cleavage sites by topo IV from drug-induced preferences. Analysis revealed strong enzyme-determined requirements for −4G, −2A and −1T bases preceding the breakage site (between −1 and +1) and enzyme-unique or degenerate determinants at −3, plus drug-specific preferences at +2/+3 and for +1 purines associated with drug intercalation. Similar cleavage rules were seen additionally at the novel V-site identified here in ColE1-derived plasmids. In concert with DNA binding data, our results provide functional evidence for DNA, enzyme and drug contributions to DNA cleavage at the gate, suggest a mechanism for DNA discrimination involving enzyme-induced DNA bending/helix distortion and cleavage complex stabilization and advance understanding of fluoroquinolones as important cleavage-enhancing therapeutics.  相似文献   

11.
12.
m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme–DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.  相似文献   

13.
Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3β may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.  相似文献   

14.
Triple helix-forming oligonucleotides covalently linked to topoisomerase I inhibitors, in particular the antitumor agent camptothecin, trigger topoisomerase I-mediated DNA cleavage selectively in the proximity of the binding site of the oligonucleotide vector. In the present study, we have performed a systematic analysis of the DNA cleavage efficiency as a function of the positioning of the camptothecin derivative, either on the 3′ or the 5′ side of the triplex, and the location of the cleavage site. A previously identified cleavage site was inserted at different positions within two triplex site-containing 59 bp duplexes. Sequence-specific DNA cleavage by topoisomerase I occurs only with triplex conjugates bearing the inhibitor at the 3′-end of the oligonucleotide and on the oligopyrimidine strand of the duplex. The lack of targeted cleavage on the 5′ side is attributed to the structural differences of the 3′ and 5′ duplex–triplex DNA junctions. The changes induced in the double helix by the triple-helical structure interfere with the action of the enzyme according to a preferred spatial organization. Camptothecin conjugates of oligonucleotides provide efficient tools to probe the organization of the topoisomerase I–DNA complex and will be useful to understand the functioning of topoisomerase I in living cells.  相似文献   

15.
The TOPRIM DXDXXG residues of type IA and II topoisomerases are involved in Mg(II) binding and the cleavage-rejoining of DNA. Mutation of the strictly conserved glycine to serine in Yersinia pestis and Escherichia coli topoisomerase I results in bacterial cell killing due to inhibition of DNA religation after DNA cleavage. In this study, all other substitutions at the TOPRIM glycine of Y. pestis topoisomerase I were examined. While the Gly to Ala substitution allowed both DNA cleavage and religation, other mutations abolished DNA cleavage. DNA cleavage activity retained by the Gly to Ser mutant could be significantly enhanced by a second mutation of the methionine residue adjacent to the active site tyrosine. Induction of mutant topoisomerase with both the TOPRIM glycine and active site region methionine mutations resulted in up to 40-fold higher cell killing rate when compared with the single TOPRIM Gly to Ser mutant. Bacterial type IA topoisomerases are potential targets for discovery of novel antibiotics. These results suggest that compounds that interact simultaneously with the TOPRIM motif and the molecular surface around the active site tyrosine could be highly efficient topoisomerase poisons through both enhancement of DNA cleavage and inhibition of DNA rejoining.  相似文献   

16.
We have investigated cauliflower (Brassica oleracea) topoisomerase II with respect to its interaction with DNA and demonstrate that the enzyme shares the characteristics of topoisomerase II purified from a variety of phylogenetically remote organisms. In the presence of the 2-nitroimidazole Ro 15-0216, cauliflower topoisomerase II-mediated DNA cleavage is extensively stimulated (approximately 20-fold) only at a site recognized as a major cleavage site for the enzyme in the absence of drug. The conservation of the enzyme's DNA specificity in the presence of Ro 15-0216 is in contrast to the effect exerted by traditional topoisomerase II inhibitors, which cause enzyme-mediated cleavage to take place at a multiple number of DNA sites. Ro 15-0216 may therefore prove useful as a tool in the elucidation of the enzyme's DNA interaction sites and its involvement in nucleic acid metabolism in plant cells.  相似文献   

17.
Abstract

A partial DNA duplex containing a high efficiency topoisomerase I cleavage site was substituted singly at each of three sites with 3′-deoxyadenosine. Depending on the site of substitution, the facility of the topoisomerase I-mediated cleavage or ligation reactions was altered. Inclusion of the modified nucleoside at the 5′-end of the acceptor oligonucleotide diminished the rate of religation following substrate cleavage by the enzyme.

  相似文献   

18.
The MvaI restriction endonuclease cuts 5′-CC↓AGG-3′/5′-CC↑TGG-3′ sites as indicated by the arrows. N4-methylation of the inner cytosines (Cm4CAGG/Cm4CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: Cm5C↓GGG/CCm5CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CCm5C↓GGG/CCm5C↑GGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the ‘normal’ activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence.  相似文献   

19.
Epipodophyllotoxins are effective antitumour drugs that trap eukaryotic DNA topoisomerase II in a covalent complex with DNA. Based on DNA cleavage assays, the mode of interaction of these drugs was proposed to involve amino acid residues of the catalytic site. An in vitro binding study, however, revealed two potential binding sites for etoposide within human DNA topoisomerase IIα (htopoIIα), one in the catalytic core of the enzyme and one in the ATP-binding N-terminal domain. Here we have tested how N-terminal mutations that reduce the affinity of the site for etoposide or ATP affect the sensitivity of yeast cells to etoposide. Surprisingly, when introduced into full-length enzymes, mutations that lower the drug binding capacity of the N-terminal domain in vitro render yeast more sensitive to epipodophyllotoxins. Consistently, when the htopoIIα N-terminal domain alone is overexpressed in the presence of yeast topoII, cells become more resistant to etoposide. Point mutations that weaken etoposide binding eliminate this resistance phenotype. We argue that the N-terminal ATP-binding pocket competes with the active site of the holoenzyme for binding etoposide both in cis and in trans with different outcomes, suggesting that each topoisomerase II monomer has two non-equivalent drug-binding sites.  相似文献   

20.
We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.  相似文献   

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