Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage

Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the Gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (−1 position). Mutagenesis of −1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable −1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the −1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.     

DNA sequences required to induce localized conversion in Streptococcus pneumoniae transformation

Summary In pneumococcal transformation a particular point mutation belonging to the amiA locus is able markedly to enhance recombination frequency when crossed with any other markers of this gene. This results from a polarized conversion of the mutation towards the wild-type sequence. In this report, by site-directed oligonucleotide mutagenesis, we have generated a series of mutants showing various degrees of conversion. We have found that the substitution 5-ATTCAT5-ATTAAT is a sufficient signal for localized conversion. Changing individual bases within this sequence results in decreased conversion frequencies to levels that depend on the mutation, suggesting that there is a family to related sequences which may act as a substrate for a conversion system. Moreover, the length over which this conversion occurs has been estimated to be 12 base pairs on the average.     

New alkaloid antibiotics that target the DNA topoisomerase I of Streptococcus pneumoniae

Streptococcus pneumoniae has two type II DNA-topoisomerases (DNA-gyrase and DNA topoisomerase IV) and a single type I enzyme (DNA-topoisomerase I, TopA), as demonstrated here. Although fluoroquinolones target type II enzymes, antibiotics efficiently targeting TopA have not yet been reported. Eighteen alkaloids (seven aporphine and 11 phenanthrenes) were semisynthesized from boldine and used to test inhibition both of TopA activity and of cell growth. Two phenanthrenes (seconeolitsine and N-methyl-seconeolitsine) effectively inhibited both TopA activity and cell growth at equivalent concentrations (～17 μM). Evidence for in vivo TopA targeting by seconeolitsine was provided by the protection of growth inhibition in a S. pneumoniae culture in which the enzyme was overproduced. Additionally, hypernegative supercoiling was observed in an internal plasmid after drug treatment. Furthermore, a model of pneumococcal TopA was made based on the crystal structure of Escherichia coli TopA. Docking calculations indicated strong interactions of the alkaloids with the nucleotide-binding site in the closed protein conformation, which correlated with their inhibitory effect. Finally, although seconeolitsine and N-methyl-seconeolitsine inhibited TopA and bacterial growth, they did not affect human cell viability. Therefore, these new alkaloids can be envisaged as new therapeutic candidates for the treatment of S. pneumoniae infections resistant to other antibiotics.     

Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site

Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/c)(A/t)A*GNNCt(T/a)N(C/a) and GN4G(G/c)(A/c)G*GNNCtTN(C/a) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.     

Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV

It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme.     

ATP-bound conformation of topoisomerase IV: a possible target for quinolones in Streptococcus pneumoniae

Topoisomerase IV, a C(2)E(2) tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.     

XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining

DNA nonhomologous end-joining (NHEJ) is a predominant pathway of DNA double-strand break repair in mammalian cells, and defects in it cause radiosensitivity at the cellular and whole-organism levels. Central to NHEJ is the protein complex containing DNA Ligase IV and XRCC4. By searching for additional XRCC4-interacting factors, we identified a previously uncharacterized 33 kDa protein, XRCC4-like factor (XLF, also named Cernunnos), that has weak sequence homology with XRCC4 and is predicted to display structural similarity to XRCC4. We show that XLF directly interacts with the XRCC4-Ligase IV complex in vitro and in vivo and that siRNA-mediated downregulation of XLF in human cell lines leads to radiosensitivity and impaired NHEJ. Furthermore, we establish that NHEJ-deficient 2BN cells derived from a radiosensitive and immune-deficient patient lack XLF due to an inactivating frameshift mutation in its gene, and that reintroduction of wild-type XLF into such cells corrects their radiosensitivity and NHEJ defects. XLF thus constitutes a novel core component of the mammalian NHEJ apparatus.     

Breakage-reunion domain of Streptococcus pneumoniae topoisomerase IV: crystal structure of a gram-positive quinolone target

The 2.7 A crystal structure of the 55-kDa N-terminal breakage-reunion domain of topoisomerase (topo) IV subunit A (ParC) from Streptococcus pneumoniae, the first for the quinolone targets from a gram-positive bacterium, has been solved and reveals a 'closed' dimer similar in fold to Escherichia coli DNA gyrase subunit A (GyrA), but distinct from the 'open' gate structure of Escherichia coli ParC. Unlike GyrA whose DNA binding groove is largely positively charged, the DNA binding site of ParC exhibits a distinct pattern of alternating positively and negatively charged regions coincident with the predicted positions of the grooves and phosphate backbone of DNA. Based on the ParC structure, a new induced-fit model for sequence-specific recognition of the gate (G) segment by ParC has been proposed. These features may account for the unique DNA recognition and quinolone targeting properties of pneumococcal type II topoisomerases compared to their gram-negative counterparts.     

The topoisomerase II poison clerocidin alkylates non-paired guanines of DNA: implications for irreversible stimulation of DNA cleavage

Clerocidin, a diterpenoid with antibacterial and antitumor activity, stimulates in vitro DNA cleavage mediated by mammalian and bacterial topoisomerase (topo) II. Different from the classical topoisomerase poisons, clerocidin-stimulated breaks at guanines immediately preceding the sites of DNA cleavage are not resealed upon heat or salt treatment. To understand the mechanism of irreversible trapping of the topo II-cleavable complex, we have investigated the reactivity of clerocidin per se towards DNA. We show here that the drug is able to nick negatively supercoiled plasmids. DNA cleavage by clerocidin in enzyme-free medium is due to the ability of the drug to form covalent adducts with guanines. Indeed, clerocidin was able to specifically react with short oligonucleotides when the guanines were unpaired and exposed as in bulges or in the single-strand form. The clerocidin epoxy group attacks the nitrogen at position 7 of guanines, leading to strand scission at the modified site. Our findings also demonstrate that trapping of topoisomerases by clerocidin is specific for type II enzymes. The guanine-alkylating ability of clerocidin suggests an unprecedented mechanism of topo II poisoning, according to which the enzyme renders the drug reactive toward DNA by distorting the double-helical structure of the nucleic acid at the cleavage site.     

Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.

DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.     

Action of quinolones against Staphylococcus aureus topoisomerase IV: basis for DNA cleavage enhancement

Topoisomerase IV is the primary cellular target for most quinolones in Gram-positive bacteria; however, its interaction with these agents is poorly understood. Therefore, the effects of four clinically relevant antibacterial quinolones (ciprofloxacin, and three new generation quinolones: trovafloxacin, levofloxacin, and sparfloxacin) on the DNA cleavage/religation reaction of Staphylococcus aureus topoisomerase IV were characterized. These quinolones stimulated enzyme-mediated DNA scission to a similar extent, but their potencies varied significantly. Drug order in the absence of ATP was trovafloxacin > ciprofloxacin > levofloxacin > sparfloxacin. Potency was enhanced by ATP, but to a different extent for each drug. Under all conditions examined, trovafloxacin was the most potent quinolone and sparfloxacin was the least. The enhanced potency of trovafloxacin correlated with several properties. Trovafloxacin induced topoisomerase IV-mediated DNA scission more rapidly than other quinolones and generated more cleavage at some sites. The most striking correlation, however, was between quinolone potency and inhibition of enzyme-mediated DNA religation: the greater the potency, the stronger the inhibition. Dose-response experiments with two topoisomerase IV mutants that confer clinical resistance to quinolones (GrlA(Ser80Phe) and GrlA(Glu84Lys)) indicate that resistance is caused by a decrease in both drug affinity and efficacy. Trovafloxacin is more active against these enzymes than ciprofloxacin because it partially overcomes the effect on affinity. Finally, comparative studies on DNA cleavage and decatenation suggest that the antibacterial properties of trovafloxacin result from increased S. aureus topoisomerase IV-mediated DNA cleavage rather than inhibition of enzyme catalysis.     

The Werner syndrome gene product co-purifies with the DNA replication complex and interacts with PCNA and topoisomerase I

Werner syndrome (WS) is a recessive disorder characterized by genomic instability and by the premature onset of a number of age-related diseases. To understand the molecular basis of this disease, we deleted a segment of the murine Wrn gene and created Wrn-deficient embryonic stem (ES) cells. At the molecular level, wild type-but not mutant-WS protein co-purifies through a series of centrifugation, chromatography, and sucrose gradient steps with the well characterized 17 S multiprotein DNA replication complex. Furthermore, wild type WS protein co-immunoprecipitates with a prominent component of the multiprotein replication complex, proliferating cell nuclear antigen (PCNA). In vitro studies also indicate that PCNA binds to a region in the N terminus portion of the WS protein containing a potential 3'-5' exonuclease domain. Finally, human WS protein also co-immunoprecipitates with both PCNA and topoisomerase I. These results suggest that the WS protein interacts with several components of the DNA replication fork.     

Protein kinase C delta activates topoisomerase IIalpha to induce apoptotic cell death in response to DNA damage

DNA topoisomerase II is an essential nuclear enzyme that modulates DNA processes by altering the topological state of double-stranded DNA. This enzyme is required for chromosome condensation and segregation; however, the regulatory mechanism of its activation is largely unknown. Here we demonstrate that topoisomerase IIalpha is activated in response to genotoxic stress. Concomitant with the activation, the expression of topoisomerase IIalpha is increased following DNA damage. The results also demonstrate that the proapoptotic kinase protein kinase C delta (PKCdelta) interacts with topoisomerase IIalpha. This association is in an S-phase-specific manner and is required for stabilization and catalytic activation of topoisomerase IIalpha in response to DNA damage. Conversely, inhibition of PKCdelta activity attenuates DNA damage-induced activation of topoisomerase IIalpha. Finally, aberrant activation of topoisomerase IIalpha by PKCdelta is associated with induction of apoptosis upon exposure to genotoxic agents. These findings indicate that PKCdelta regulates topoisomerase IIalpha and thereby cell fate in the genotoxic stress response.     

Mutational analysis of Escherichia coli topoisomerase IV. II. ATPase negative mutants of parE induce hyper-DNA cleavage

ParE is the ATP-binding subunit of topoisomerase IV (Topo IV). During topoisomerization, the ATP-binding and hydrolysis cycle must be coordinated with the cycle of DNA cleavage and religation. We have isolated three dominant-negative mutant alleles of parE that encode ParE proteins that fail to hydrolyze ATP when reconstituted with ParC to form Topo IV. ParE G110S Topo IV and ParE S123L Topo IV failed to bind ATP at all, whereas ParE T201A could bind ATP. All three mutant Topo IV proteins exhibited an elevated level of spontaneous DNA cleavage that could be associated with a decreased rate of DNA resealing. In ParE T201A Topo IV, this defect appeared to result from an increased likelihood that the tetrameric enzyme would fall apart after DNA cleavage. Thus, while ATP is not required for DNA cleavage, the properties of these mutant enzymes suggests that ATP-hydrolysis informs DNA religation.     

The DNA damage response mediator MDC1 directly interacts with the anaphase-promoting complex/cyclosome

MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (gamma-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of gamma-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.     

Transfection of Streptococcus pneumoniae with bacteriophage DNA.

It was possible to transfect Streptococcus pneumoniae with DNA obtained from a newly isolated bacteriophage, diplophage-4 (Dp-4). Optimal frequency of transfection (0.9%) required the use of a nuclease-defective mutant; with wild-type bacteria, the transfection frequency was about 100-fold lower. Transfection requires physiological conditions that appear to be similar to the competent state needed for genetic transformation (A. Tomasz, J. Bacteriol. 91:1050--1061, 1966).     

Fate of DNA in eclipse complex during genetic transformation in Streptococcus pneumoniae

Uptake of DNA and genetic recombination proceeded normally in competent Streptococcus pneumoniae despite inhibition of DNA replication by 6-(p-hydroxyphenylazo)-uracil. Immediately after a brief uptake period, 68% of donor DNA label was in eclipse complex form, and 22% was in low-molecular-weight products; by the completion of integration at 10 min, 23% was integrated into the chromosome, and the rest was lost from the cell. Throughout the process, less than 1% was found as free single strands. The DNA in eclipse complex is therefore an intermediate in the integration process.     

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.     

The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae

Background

Streptococcus pneumoniae is the major cause of community-acquired pneumonia and is also associated with bronchitis, meningitis, otitis and sinusitis. The emergence and increasing prevalence of resistance to penicillin and other antibiotics has led to interest in other anti-pneumonococcal drugs such as quinolones that target the enzymes DNA gyrase and topoisomerase IV. During crystallization and in the avenues to finding a method to determine phases for the structure of the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae, obstacles were faced at each stage of the process. These problems included: majority of the crystals being twinned, either non-diffracting or exhibiting a high mosaic spread. The crystals, which were grown under conditions that favoured diffraction, were difficult to flash-freeze without loosing diffraction. The initial structure solution by molecular replacement failed and the approach proved to be unviable due to the complexity of the problem. In the end the successful structure solution required an in-depth data analysis and a very detailed molecular replacement search.

Methodology/Principal Findings

Crystal anti-twinning agents have been tested and two different methods of flash freezing have been compared. The fragility of the crystals did not allow the usual method of transferring the crystals into the heavy atom solution. Consequently, it was necessary to co-crystallize in the presence of the heavy atom compound. The multiple isomorphous replacement approach was unsuccessful because the 7 cysteine mutants which were engineered could not be successfully derivatized. Ultimately, molecular replacement was used to solve the structure by sorting through a large number of solutions in space group P1 using CNS.

Conclusions/Significance

The main objective of this paper is to describe the obstacles which were faced and overcome in order to acquire data sets on such difficult crystals and determine phases for successful structure solution.