Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata

A type II DNA topoisomerase (topollmt), purified to near homogeneity from the trypanosomatid C. fasciculata has been shown to be localized to the single mitochondrion of these kinetoplastid protozoa. Immunoblots show at least a 10-fold higher level of topollmt (per milligram of protein) in preparations of partially purified mitochondria as compared with those from whole cells. Analyses of type I and type II topoisomerase activities in both mitochondrial and whole cell extracts show a 4- to 5-fold higher specific activity of topollmt in mitochondrial extracts while a nuclear type I topoisomerase has a 4- to 5-fold lower specific activity in the same extract. Immunolocalizations using anti-topollmt antibodies show the enzyme to be present in close association with the mitochondrial DNA networks (kinetoplast DNA or kDNA). This association appears at two distinct locations on opposite sides of the kDNA network.     

Characterization of a unique type IA topoisomerase in Bacillus cereus

Bacillus cereus topoisomerase IIIbeta (bcTopo IIIbeta) has been cloned, overexpressed and biochemically characterized. This enzyme exhibits 64% and 33% sequence identity to Bacillus subtilis topoisomerase III (bsTopo III) and Escherichia coli topoisomerase III (ecTopo III) respectively. The enzymatic properties of bcTopo IIIbeta differ substantially from other bacterial type IA topoisomerases, including E. coli type IA topoisomerases and B. cereus topoisomerase I (bcTopo I) and IIIalpha (bcTopo IIIalpha). bcTopo IIIbeta only partially relaxes negatively supercoiled DNA and appears incapable of generating fully relaxed topoisomers. In contrast to ecTopo III and bcTopo IIIalpha, bcTopo IIIbeta is not a decatenase. bcTopo IIIbeta is unable to compensate the loss of ecTopo III in vivo. Therefore, bcTopo IIIbeta is a unique prokaryotic type IA topoisomerase that is different from previously characterized topoisomerases.     

Distinct genes encode type II Topoisomerases for the nucleus and mitochondrion in the protozoan parasite Trypanosoma brucei

Topoisomerases are essential for orderly nucleic acid metabolism and cell survival and are proven targets for clinically useful antimicrobial and anticancer drugs. Interest in the topologically intricate mitochondrial DNA (kinetoplast or kDNA) of Trypanosoma brucei brucei and related kinetoplastid protozoan parasites has led to many reports of type II topoisomerases that participate in kDNA metabolism (we term the T. brucei brucei gene TbTOP2mt). We have now identified and characterized two new genes for type II topoisomerases in T. brucei brucei, termed TbTOP2alpha and TbTOP2beta. Phylogenetically, they share a common node with other nuclear topoisomerases, clearly distinct from a clade that includes the previously reported kinetoplastid genes, all of which are homologs of TbTOP2mt. Southern blot analysis reveals the new genes are single copy and positioned approximately 1.7 kb apart. Cognate mRNAs are expressed in African trypanosomes, but only a single message is detected in Leishmania or Crithidia. TbTOP2alpha encodes an ATP-dependent topoisomerase that appears as a single approximately 170-kDa band on immunoblots and localizes to the nucleus; RNA interference leads to pleomorphic nuclear (but not kDNA) abnormalities and early growth arrest. The role of TbTOP2beta is unclear. Although transcribed in trypanosomes, TbTOP2beta is not detected by beta-specific antiserum, and RNAi silencing results in no obvious phenotype. These studies indicate that African trypanosomes and related kinetoplastid human pathogens are unusual in having independent topoisomerase II genes to service their nuclear and mitochondrial genomes, and they highlight TbTOP2alpha as a promising target for the development of much-needed new therapies.     

Reverse gyrase recruitment to DNA after UV light irradiation in Sulfolobus solfataricus

Induction of DNA damage triggers a complex biological response concerning not only repair systems but also virtually every cell function. DNA topoisomerases regulate the level of DNA supercoiling in all DNA transactions. Reverse gyrase is a peculiar DNA topoisomerase, specific to hyperthermophilic microorganisms, which contains a helicase and a topoisomerase IA domain that has the unique ability to introduce positive supercoiling into DNA molecules. We show here that reverse gyrase of the archaean Sulfolobus solfataricus is mobilized to DNA in vivo after UV irradiation. The enzyme, either purified or in cell extracts, forms stable covalent complexes with UV-damaged DNA in vitro. We also show that the reverse gyrase translocation to DNA in vivo and the stabilization of covalent complexes in vitro are specific effects of UV light irradiation and do not occur with the intercalating agent actinomycin D. Our results suggest that reverse gyrase might participate, directly or indirectly, in the cell response to UV light-induced DNA damage. This is the first direct evidence of the recruitment of a topoisomerase IA enzyme to DNA after the induction of DNA damage. The interaction between helicase and topoisomerase activities has been previously proposed to facilitate aspects of DNA replication or recombination in both Bacteria and Eukarya. Our results suggest a general role of the association of such activities in maintaining genome integrity and a mutual effect of DNA topology and repair.     

Bacterial cell killing mediated by topoisomerase I DNA cleavage activity

DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerase can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such a stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS-inducing and cell-killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents.     

Helicase-appended Topoisomerases: New Insight into the Mechanism of Directional Strand Transfer

DNA strand passage through an enzyme-mediated gate is a key step in the catalytic cycle of topoisomerases to produce topological transformations in DNA. In most of the reactions catalyzed by topoisomerases, strand passage is not directional; thus, the enzyme simply provides a transient DNA gate through which DNA transport is allowed and thereby resolves the topological entanglement. When studied in isolation, the type IA topoisomerase family appears to conform to this rule. Interestingly, type IA enzymes can carry out directional strand transport as well. We examined here the biochemical mechanism for directional strand passage of two type IA topoisomerases: reverse gyrase and a protein complex of topoisomerase IIIα and Bloom helicase. These enzymes are able to generate vectorial strand transport independent of the supercoiling energy stored in the DNA molecule. Reverse gyrase is able to anneal single strands, thereby increasing linkage number of a DNA molecule. However, topoisomerase IIIα and Bloom helicase can dissolve DNA conjoined with a double Holliday junction, thus reducing DNA linkage. We propose here that the helicase or helicase-like component plays a determinant role in the directionality of strand transport. There is thus a common biochemical ground for the directional strand passage for the type IA topoisomerases.     

Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.     

Drosophila topoisomerase II-DNA interactions are affected by DNA structure.

The binding of purified Drosophila topoisomerase II to the highly bent DNA segments from the SV40 terminus of replication and C. fasciculata kinetoplast minicircle DNA (kDNA) was examined using electron microscopy (EM). The probability of finding topoisomerase II positioned at or near the bent SV40 terminus and Crithidia fasciculata kDNA was two- and threefold higher, respectively, than along the unbent pBR325 DNA into which the elements had been cloned. Closer examination demonstrated that the enzyme bound preferentially to the junction between the bent and non-bent sequences. Using gel electrophoresis, a cluster of strong sodium dodecyl sulfate-induced topoisomerase II cleavage sites was mapped to the SV40 terminus DNA, and two weak cleavage sites to the C. fasciculata kDNA. As determined by EM, Drosophila topoisomerase II foreshortened the apparent length of DNA by only 15 base-pairs when bound, arguing that it does not wrap DNA around itself. When bound to pBR325 containing the C. fasciculata kDNA and the SV40 terminus, topoisomerase II often produced DNA loops. The size distribution was that predicted from the known probability of any two points along linear DNA colliding. In vitro mapping of topoisomerase II on DNA whose ends were blocked by avidin protein revealed that binding is enhanced at sites located near a blocked end as compared to a free end. These observations may contribute towards establishing a framework for understanding topoisomerase II-DNA interactions.     

RNA interference of a trypanosome topoisomerase II causes progressive loss of mitochondrial DNA

We studied the function of a Trypanosoma brucei topoisomerase II using RNA interference (RNAi). Expression of a topoisomerase II double-stranded RNA as a stem-loop caused specific degradation of mRNA followed by loss of protein. After 6 days of RNAi, the parasites' growth rate declined and the cells subsequently died. The most striking phenotype upon induction of RNAi was the loss of kinetoplast DNA (kDNA), the cell's catenated mitochondrial DNA network. The loss of kDNA was preceded by gradual shrinkage of the network and accumulation of gapped free minicircle replication intermediates. These facts, together with the localization of the enzyme in two antipodal sites flanking the kDNA, show that a function of this topoisomerase II is to attach free minicircles to the network periphery following their replication.     

Flexibility at Gly-194 is required for DNA cleavage and relaxation activity of Escherichia coli DNA topoisomerase I

The proposed mechanism of type IA DNA topoisomerase I includes conformational changes by the single enzyme polypeptide to allow binding of the G strand of the DNA substrate at the active site, and the opening or closing of the "gate" created on the G strand of DNA to the passing single or double DNA strand(s) through the cleaved G strand DNA. The shifting of an alpha helix upon G strand DNA binding has been observed from the comparison of the type IA DNA topoisomerase crystal structures. Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this alpha helix in Escherichia coli DNA topoisomerase I showed that flexibility around this glycine residue is required for DNA cleavage and relaxation activity and supports a functional role for this hinge region in the enzyme conformational change.     

A highly processive topoisomerase I: studies at the single-molecule level

Amongst enzymes which relieve torsional strain and maintain chromosome supercoiling, type IA topoisomerases share a strand-passage mechanism that involves transient nicking and re-joining of a single deoxyribonucleic acid (DNA) strand. In contrast to many bacterial species that possess two type IA topoisomerases (TopA and TopB), Actinobacteria possess only TopA, and unlike its homologues this topoisomerase has a unique C-terminal domain that lacks the Zn-finger motifs characteristic of type IA enzymes. To better understand how this unique C-terminal domain affects the enzyme''s activity, we have examined DNA relaxation by actinobacterial TopA from Streptomyces coelicolor (ScTopA) using real-time single-molecule experiments. These studies reveal extremely high processivity of ScTopA not described previously for any other topoisomerase of type I. Moreover, we also demonstrate that enzyme processivity varies in a torque-dependent manner. Based on the analysis of the C-terminally truncated ScTopA mutants, we propose that high processivity of the enzyme is associated with the presence of a stretch of positively charged amino acids in its C-terminal region.     

Structural studies of E. coli topoisomerase III-DNA complexes reveal a novel type IA topoisomerase-DNA conformational intermediate

Escherichia coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5' phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an eight-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is bound along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding.     

Metal ion and inter-domain interactions as functional networks in E. coli topoisomerase I

Escherichia coli topoisomerase I (EcTopoI) is a type IA bacterial topoisomerase which is receiving large attention due to its potential application as novel target for antibacterial therapeutics. Nevertheless, a detailed knowledge of its mechanism of action at molecular level is to some extent lacking. This is partly due to the requirement of several factors (metal ions, nucleic acid) to the proper progress of the enzyme catalytic cycle. Additionally, each of them can differently affect the protein structure.     

Mitochondrial DNA polymerase POLIB is essential for minicircle DNA replication in African trypanosomes

The unique mitochondrial DNA of trypanosomes is a catenated network of minicircles and maxicircles called kinetoplast DNA (kDNA). The network is essential for survival, and requires an elaborate topoisomerase‐mediated release and reattachment mechanism for minicircle theta structure replication. At least seven DNA polymerases (pols) are involved in kDNA transactions, including three essential proteins related to bacterial DNA pol I (POLIB, POLIC and POLID). How Trypanosoma brucei utilizes multiple DNA pols to complete the topologically complex task of kDNA replication is unknown. To fill this gap in knowledge we investigated the cellular role of POLIB using RNA interference (RNAi). POLIB silencing resulted in growth inhibition and progressive loss of kDNA networks. Additionally, unreplicated covalently closed precursors become the most abundant minicircle replication intermediate as minicircle copy number declines. Leading and lagging strand minicircle progeny similarly declined during POLIB silencing, indicating POLIB had no apparent strand preference. Interestingly, POLIB RNAi led to the accumulation of a novel population of free minicircles that is composed mainly of covalently closed minicircle dimers. Based on these data, we propose that POLIB performs an essential role at the core of the minicircle replication machinery.     

Cloning and biochemical characterization of Staphylococcus aureus type IA DNA topoisomerase comprised of distinct five domains

DNA topoisomerases play critical roles in regulating DNA topology and are essential enzymes for cell survival. In this study, a gene encoding type IA DNA topoisomerase was cloned from Staphylococcus aureus (S. aureus) sp. strain C-66, and the biochemical properties of recombinant enzyme was characterized. The nucleotide sequence analysis showed that the cloned gene contained an open reading frame (2070 bp) that could encode a polypeptide of 689 amino acids. The cloned gene actually produced 79.1 kDa functional enzyme (named Sau-TopoI) in Escherichia coli (E. coli). Sau-TopoI enzyme purified from E. coli showed ATP-independent and Mg²⁺-dependent manners for relaxing negatively supercoiled DNA. The relaxation activity of Sau-TopoI was inhibited by camptothecin, but not by nalidixic acid and etoposide. Cleavage site mapping showed that the enzyme could preferentially bind to and cleave the sequence GGNN↓CAT (N and ↓ represent any nucleotide and cleavage site, respectively). All these results suggest that the purified enzyme is type IA DNA topoisomerase. In addition, domain mapping analysis showed that the enzyme was composed of conserved four domains (I through IV), together with a variable C-terminal region containing a unique domain V.     

Inhibition of Zn(II) Binding Type IA Topoisomerases by Organomercury Compounds and Hg(II)

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.