Tryptophane-205 of human topoisomerase I is essential for camptothecin inhibition of negative but not positive supercoil removal

Positive supercoils are introduced in cellular DNA in front of and negative supercoils behind tracking polymerases. Since DNA purified from cells is normally under-wound, most studies addressing the relaxation activity of topoisomerase I have utilized negatively supercoiled plasmids. The present report compares the relaxation activity of human topoisomerase I variants on plasmids containing equal numbers of superhelical twists with opposite handedness. We demonstrate that the wild-type enzyme and mutants lacking amino acids 1–206 or 191–206, or having tryptophane-205 replaced with a glycine relax positive supercoils faster than negative supercoils under both processive and distributive conditions. In contrast to wild-type topoisomerase I, which exhibited camptothecin sensitivity during relaxation of both negative and positive supercoils, the investigated N-terminally mutated variants were sensitive to camptothecin only during removal of positive supercoils. These data suggest different mechanisms of action during removal of supercoils of opposite handedness and are consistent with a recently published simulation study [Sari and Andricioaei (2005) Nucleic Acids Res., 33, 6621–6634] suggesting flexibility in distinct parts of the enzyme during clockwise or counterclockwise strand rotation.     

High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius

A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 - 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine-5' -0-(3-thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP-driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.=     

Negative constrained DNA supercoiling in archaeal nucleosomes

Archaeal histones have significant sequence and structural similarity to their eukaryal counterparts. However, whereas DNA is wrapped in negatively constrained supercoils in eukaryal nucleosomes, it has been reported that DNA is positively supercoiled by archaeal nucleosomes. This was inferred from experiments performed at low temperature and low salt concentrations, conditions markedly different from those expected for many archaea in vivo. Here, we report that the archaeal histones HMf and HTz wrap DNA in negatively constrained supercoils in buffers containing potassium glutamate (K-Glu) above 300 mM, either at 37 degrees C or at 70 degrees C. This suggests that high salt concentrations allow an alternate archaeal nucleosome topology: a left-handed tetramer rather than the right-handed tetramer seen in low salt conditions. In contrast, the archaeal histone MkaH produces DNA negative supercoiling at all salt concentrations, suggesting that this duality of structure is not possible for this atypical protein, which is formed by the association of two histone folds in a single polypeptide. These results extend the already remarkable similarity between archaeal and eukaryal nucleosomes, as it has been recently shown that DNA can be wrapped into either positive or negative supercoils around the H3/H4 tetramer. Negative supercoiling could correspond to the predominant physiological mode of DNA supercoiling in archaeal nucleosomes.     

Human topoisomerase IIalpha rapidly relaxes positively supercoiled DNA: implications for enzyme action ahead of replication forks

Movement of the DNA replication machinery through the double helix induces acute positive supercoiling ahead of the fork and precatenanes behind it. Because topoisomerase I and II create transient single- and double-stranded DNA breaks, respectively, it has been assumed that type I enzymes relax the positive supercoils that precede the replication fork. Conversely, type II enzymes primarily resolve the precatenanes and untangle catenated daughter chromosomes. However, studies on yeast and bacteria suggest that type II topoisomerases may also function ahead of the replication machinery. If this is the case, then positive DNA supercoils should be the preferred relaxation substrate for topoisomerase IIalpha, the enzyme isoform involved in replicative processes in humans. Results indicate that human topoisomerase IIalpha relaxes positively supercoiled plasmids >10-fold faster than negatively supercoiled molecules. In contrast, topoisomerase IIbeta, which is not required for DNA replication, displays no such preference. In addition to its high rates of relaxation, topoisomerase IIalpha maintains lower levels of DNA cleavage complexes with positively supercoiled molecules. These properties suggest that human topoisomerase IIalpha has the potential to alleviate torsional stress ahead of replication forks in an efficient and safe manner.     

Large scale preparation of positively supercoiled DNA using the archaeal histone HMf.

A technique to prepare relatively large quantities (>/=100 microg) of highly positively supercoiled DNA is reported. This uses a recombinant archaeal histone (rHMfB) to introduce toroidal supercoils, and an inexpensive chicken blood extract to relax unrestrained superhelical tension. Preparation of positively supercoiled pUC19 DNA molecules, >50% of which have linking number changes ranging from+8 to+17, is demonstrated. Advantages include the high degree of positive supercoiling that can be achieved, control over the extent of supercoiling, easy production of relatively large quantities of supercoiled DNA, and low cost.     

DNA-loop formation on nucleosomes shown by in situ scanning force microscopy of supercoiled DNA

The flexibility of the chromatin structure, necessary for the processing of the genomic DNA, is controlled by a number of factors where flexibility and mobility of the nucleosomes is essential. Here, the influence of DNA supercoiling on the structure of single nucleosomes is investigated. Circular supercoiled plasmid DNA sub-saturated with histones was visualized by scanning force microscopy (SFM) in aqueous solution. SFM-imaging compared with topological analysis indicates instability of nucleosomes when the salt concentration is raised from 10 mM to 100 mM NaCl. Nucleosomes were observed after the deposition to the used scanning surface, i.e. mica coated with polylysine. On the images, the nucleosomes appear with a high probability in end-loops near the apices of the superhelices. In 100 mM NaCl but not in 10 mM NaCl, a significant number of complexes present the nucleosomes on superhelical crossings mainly located adjacent to an end-loop. The morphology of these structures and statistical analysis suggest that DNA loops were formed on the histone octamers, where the loop size distribution shows a pronounced peak at 50 nm. Recently, the formation and diffusion of loops on octamers has been discussed as a mechanism of translocations of nucleosomes along DNA. The presented data likely confirm the occurrence of loops, which may be stabilized by supercoiling. Analysis of the structure of regular nucleosomes not located on crossings indicates that reducing the salt concentration leads to more conformations, where DNA is partially unwrapped from the distal ends of the octamer.     

The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as ‘topological poisons of topoisomerase I’.     

DNA gyrase can supercoil DNA circles as small as 174 base pairs.

DNA gyrase introduces negative supercoils into closed-circular DNA using the free energy of ATP hydrolysis. Consideration of steric and thermodynamic aspects of the supercoiling reaction indicates that there should be a lower limit to the size of DNA circle which can be supercoiled by gyrase. We have investigated the supercoiling reaction of circles from 116-427 base pairs (bp) in size and have determined that gyrase can supercoil certain relaxed isomers of circles as small as 174 bp, dependent on the final superhelix density of the supercoiled product. Furthermore, this limiting superhelical density (-0.11) is the same as that determined for the supercoiling of plasmid pBR322. We also find that although circles in the range 116-152 bp cannot be supercoiled, they can nevertheless be relaxed by gyrase when positively supercoiled. These data suggest that the conformational changes associated with the supercoiling reaction can be carried out by gyrase in a circle as small as 116 bp. We discuss these results with respect to the thermodynamics of DNA supercoiling and steric aspects of the gyrase mechanism.