Analysis of amplified DNAs from drug-resistant Leishmania by orthogonal-field-alternation gel electrophoresis. The effect of size and topology on mobility

Amplified extrachromosomal DNAs from antifolate-resistant Leishmania are 30-75 kilobase (kb) supercoiled molecules that resolve on orthogonal-field-alternation gel electrophoresis (OFAGE) gels. These DNAs comigrate with smaller supercoiled plasmids (7-8 kb), and their mobility is not a simple function of their size. The properties of the amplified DNAs were investigated to determine if an unusual structure accounts for the observed mobility of the amplified DNAs by OFAGE; however, their topological properties were similar to those of standard Escherichia coli plasmids. The migration of a series of supercoiled plasmids ranging in size from 6 to 91 kb was analyzed by OFAGE, and a triphasic pattern was observed. The mobilities of plasmids between 20 and 60 kb increase with size, whereas the migration of plasmids between 6 and 20 and 60 and 91 kb is inversely proportional to size. Like smaller plasmids, the large supercoiled DNAs show a pulse time-independent mobility by OFAGE. The mobility of amplified DNA from Leishmania is in accord with that of the plasmid markers. Therefore, it is primarily the size of the amplified extrachromosomal DNAs from Leishmania, rather than an unusual superhelical density or topological structure, that results in the previously unexplained migration pattern.     

Effect of supercoiling on the juxtaposition and relative orientation of DNA sites.

There are many proteins that interact simultaneously with two or more DNA sites that are separated along the DNA contour. These sites must be brought close together to form productive complexes with the proteins. We used Monte Carlo simulation of supercoiled DNA conformations to study the effect of supercoiling and DNA length on the juxtaposition of DNA sites, the angle between them, and the branching of the interwound superhelix. Branching decreases the probability of juxtaposition of two DNA sites but increases the probability of juxtaposition of three sites at branch points. We found that the number of superhelix branches increases linearly with the length of DNA from 3 to 20 kb. The simulations showed that for all contour distances between two sites, the juxtaposition probability in supercoiled DNA is two orders of magnitude higher than in relaxed DNA. Supercoiling also results in a strong asymmetry of the angular distribution of juxtaposed sites. The effect of supercoiling on site-specific recombination and the introduction of supercoils by DNA gyrase is discussed in the context of the simulation results.     

Disentangling DNA during replication: a tale of two strands

The seminal papers by Watson and Crick in 1953 on the structure and function of DNA clearly enunciated the challenge their model presented of how the intertwined strands of DNA are unwound and separated for replication to occur. We first give a historical overview of the major discoveries in the past 50 years that address this challenge. We then describe in more detail the cellular mechanisms responsible for the unlinking of DNA. No single strategy on its own accounts for the complete unlinking of chromosomes required for DNA segregation to proceed. Rather, it is the combined effects of topoisomerase action, chromosome organization and DNA-condensing proteins that allow the successful partitioning of chromosomes into dividing cells. Finally, we propose a model of chromosome structure, consistent with recent findings, that explains how the problem of unlinking is alleviated by the division of chromosomal DNA into manageably sized domains.     

The Saccharomyces cerevisiae Smc2/4 condensin compacts DNA into (+) chiral structures without net supercoiling

Smc2/4 forms the core of the Saccharomyces cerevisiae condensin, which promotes metaphase chromosome compaction. To understand how condensin manipulates DNA, we used two in vitro assays to study the role of SMC (structural maintenance of chromosome) proteins and ATP in reconfiguring the path of DNA. The first assay evaluated the topology of knots formed in the presence of topoisomerase II. Unexpectedly, both wild-type Smc2/4 and an ATPase mutant promoted (+) chiral knotting of nicked plasmids, revealing that ATP hydrolysis and the non-SMC condensins are not required to compact DNA chirally. The second assay measured Smc2/4-dependent changes in linking number (Lk). Smc2/4 did not induce (+) supercoiling, but instead induced broadening of topoisomer distributions in a cooperative manner without altering Lk(0). To explain chiral knotting in substrates devoid of chiral supercoiling, we propose that Smc2/4 directs chiral DNA compaction by constraining the duplex to retrace its own path. In this highly cooperative process, both (+) and (-) loops are sequestered (about one per kb), leaving net writhe and twist unchanged while broadening Lk. We have developed a quantitative theory to account for these results. Additionally, we have shown at higher molar stoichiometries that Smc2/4 prevents relaxation by topoisomerase I and nick closure by DNA ligase, indicating that Smc2/4 can saturate DNA. By electron microscopy of Smc2/4-DNA complexes, we observed primarily two protein-laden bound species: long flexible filaments and uniform rings or "doughnuts." Close packing of Smc2/4 on DNA explains the substrate protection we observed. Our results support the hypothesis that SMC proteins bind multiple DNA duplexes.     

Conformational and thermodynamic properties of supercoiled DNA.

We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, superhelix axis length and the number of superhelical turns. The computed number of superhelix branches was found to be dependent on superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low superhelix density the entropic contribution to superhelix free energy is negligible, whereas at high superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.     

The effect of ionic conditions on DNA helical repeat, effective diameter and free energy of supercoiling.

We determined the free energy of DNA supercoiling as a function of the concentration of magnesium and sodium chloride in solution by measuring the variance of the equilibrium distribution of DNA linking number,<(DeltaLk)2>. We found that the free energy of supercoiling changed >1.5-fold over the range of ionic conditions studied. Comparison of the experimental results with those of computer simulations showed that the ionic condition dependence of<(DeltaLk)2>is due mostly to the change in DNA effective diameter, d, a parameter characterizing the electrostatic interaction of DNA segments. To make this comparison we determined values of d under all ionic conditions studied by measuring the probability of knot formation during random cyclization of linear DNA molecules. From the topoisomer distributions we could also determine the changes in DNA helical repeat, gamma, in mixed NaCl/MgCl2 solutions. Both gamma and d exhibited a complex pattern of changes with changing ionic conditions, which can be described in terms of competition between magnesium and sodium ions for binding to DNA.     

Altered Deoxyribonucleic Acid Polymerase Activity in a Methyl Methanesulfonate-Sensitive Mutant of Bacillus subtilis

Of six deoxyribonucleic acid repair mutants of Bacillus subtilis assayed for deoxyribonucleic acid polymerase, only the methyl methanesulfonate-sensitive and ultraviolet light-sensitive mutant JB1-49(59) has impaired polymerase activity. Extracts prepared by sonic treatment or gentle lysis had about 10% of the wild-type activity with poly d(A-T), an alternating copolymer of deoxyadenylate and deoxythymidylate, used as template. The sensitivity to methyl methanesulfonate and ultraviolet light and the low level of polymerase activity transformed and reverted together, indicating that the two characteristics are a pleiotropic manifestation of a single mutation. Mixed extract and kinetic experiments mitigated against an altered nuclease activity as the enzymatic consequence of the mutation. Also, the mutant and wild type activities were stimulated equally by Escherichia coli exonuclease III. The residual activity in the mutant showed several differences from the wild-type activity: it purified differently, was more sensitive to sulfhydryl reagents, and displayed a different template specificity. We tentatively conclude that either the mutation in JB1-49(59) has introduced a qualitative as well as a quantitative change in the polymerase or the wild type contains two distinct polymerases, one of which is missing in the mutant.     

Alteration of Escherichia coli topoisomerase IV conformation upon enzyme binding to positively supercoiled DNA

Escherichia coli topoisomerase IV (topo IV) is an essential enzyme that unlinks the daughter chromosomes for proper segregation at cell division. In vitro, topo IV readily distinguishes between the two possible chiralities of crossing segments in a DNA substrate. The enzyme relaxes positive supercoils and left-handed braids 20 times faster, and with greater processivity, than negative supercoils and right-handed braids. Here, we used chemical cross-linking of topo IV to demonstrate that enzyme bound to positively supercoiled DNA is in a different conformation from that bound to other forms of DNA. Using three different reagents, we observed novel cross-linked species of topo IV when positively supercoiled DNA was in the reaction. We show that the ParE subunits are in close enough proximity to be cross-linked only when the enzyme is bound to positively supercoiled DNA. We suggest that the altered conformation reflects efficient binding by topo IV of the two DNA segments that participate in the strand passage reaction.     

SV40 large T antigen hexamer structure: domain organization and DNA-induced conformational changes

Simian Virus 40 replication requires only one viral protein, the Large T antigen (T-ag), which acts as both an initiator of replication and as a replicative helicase (reviewed in ). We used electron microscopy to generate a three-dimensional reconstruction of the T-ag hexameric ring in the presence and absence of a synthetic replication fork to locate the T-ag domains, to examine structural changes in the T-ag hexamer associated with DNA binding, and to analyze the formation of double hexamers on and off DNA. We found that binding DNA to the T-ag hexamer induces large conformational changes in the N- and C-terminal domains of T-ag. Additionally, we observed a significant increase in density throughout the central channel of the hexameric ring upon DNA binding. We conclude that conformational changes in the T-ag hexamer are required to accommodate DNA and that the mode of DNA binding may be similar to that suggested for some other ring helicases. We also identified two conformations of T-ag double hexamers formed in the presence of forked DNA: with N-terminal hexamer-hexamer contacts, similar to those formed on origin DNA, or with C-terminal contacts, which are unlike any T-ag double hexamers reported previously.