The Bacillus subtilis primosomal protein DnaD untwists supercoiled DNA

The essential Bacillus subtilis DnaD and DnaB proteins have been implicated in the initiation of DNA replication. Recently, DNA remodeling activities associated with both proteins were discovered that could provide a link between global or local nucleoid remodeling and initiation of replication. DnaD forms scaffolds and opens up supercoiled plasmids without nicking to form open circular complexes, while DnaB acts as a lateral compaction protein. Here we show that DnaD-mediated opening of supercoiled plasmids is accompanied by significant untwisting of DNA. The net result is the conversion of writhe (Wr) into negative twist (Tw), thus maintaining the linking number (Lk) constant. These changes in supercoiling will reduce the considerable energy required to open up closed circular plectonemic DNA and may be significant in the priming of DNA replication. By comparison, DnaB does not affect significantly the supercoiling of plasmids. Binding of the DnaD C-terminal domain (Cd) to DNA is not sufficient to convert Wr into negative Tw, implying that the formation of scaffolds is essential for duplex untwisting. Overall, our data suggest that the topological effects of the two proteins on supercoiled DNA are different; DnaD opens up, untwists and converts plectonemic DNA to a more paranemic form, whereas DnaB does not affect supercoiling significantly and condenses DNA only via its lateral compaction activity. The significance of these findings in the initiation of DNA replication is discussed.     

Energetics of DNA twisting. II. Topoisomer analysis

A gel electrophoresis method has been developed for resolving small (approximately equal to 250 bp DNA topoisomers. In this size range only one major topoisomer band is observed, except for ligase closure conditions in which the probabilities are nearly equal for circularization by untwisting and overtwisting the corresponding linear DNA. The two probabilities are nearly equal when delta Tw is close to 0.5, if the mean helical twist of the linear DNA is n + delta Tw, where n is an integer and delta Tw is the fractional twist. We determine delta Tw of the linear DNA in standard conditions (20 degrees C, no ethidium) by titration experiments in which delta Tw is varied at the time of ligase closure, either by changing temperature or ethidium concentration. The endpoint (delta Tw = 0.5) is found when the two topoisomers formed by untwisting and overtwisting are present at equal concentrations. This analysis assumes that the net writhe is zero and the DNA helix is isotropically bendable. The results confirm the analysis of cyclization probabilities given in the preceding paper: delta Tw = 0 at the two maxima in the curve of j-factor versus DNA length and delta Tw = 0.5 at the minimum. Consequently, we can determine the DNA lengths at which Tw takes on integral values and use them to measure precisely the average helix repeat. From the difference between the delta Tw values of DNAs with 237 and 247 bp, we obtain an approximate value for the helix repeat of h = 10.4 +/- 0.1 bp/turn, in good agreement with earlier values found by the band-shift and nuclease-cutting methods. The twist is integral at 250.8 +/- 0.4 bp and from h = 10.4 +/- 0.1 we find n = 24; then 250.8/24 gives h = 10.45 +/- 0.02 bp/turn. The mean linking number (Lk) changes in a stepwise manner as delta Tw is varied for 250 bp DNAs. This result is expected when the free energy of twisting half a turn becomes large compared to thermal fluctuations. In these experiments, it is possible to obtain the mean Tw value from the mean Lk value only when delta Tw = 0.5, and consequently the mean Lk value is not simply related to DNA length for 250 bp DNAs except when delta Tw = 0.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Statistical mechanics of DNA topoisomers. The helical worm-like chain

Recent experimental data of Shore & Baldwin (1983b) and of Horowitz & Wang (1984) for the apparent twisting coefficient K, which determines the breadth of the Gaussian distribution of DNA topoisomers with different linking numbers N, show that the product of K and nbp (the number of base-pairs) is nearly a constant for nbp approximately greater than 2000, but that it increases sharply with decreasing nbp for nbp approximately less than 2000. The main purpose of the present paper is to explain theoretically such behavior of K as a function of nbp. Thus the statistical mechanics of DNA topoisomers in general is developed on the basis of a twisted worm-like chain, i.e. a special case of the helical worm-like chain. The previous treatments of the N-dependent ring-closure probability, i.e. the distribution of N, which are valid only for small chain length L, are extended to the range of larger L. The variance of N is then shown to be exactly the sum of those of the writhe Wr and the twist Tw. For small values of L, the distribution of Wr is not Gaussian, and its variance or moment (Wr2) increases rather steeply with increasing L. With these and known Monte Carlo results for freely jointed chains, an empirical interpolation formula for (Wr2) is also constructed to be valid for all values of L. It predicts that (Wr2)/L increases monotonically, with increasing L to its coil-limiting value. On the other hand, the distribution of N is actually Gaussian in the practical range of N for all values of L. The conditional distribution of Wr with N fixed is also evaluated. Finally, K is expressed in terms of the torsional constant C, the stiffness parameter lambda-1 (which is equal to the Kuhn segment length and twice the persistence length for this special case), and (Wr2). The derived equation predicts that nbpK decreases monotonically to its coil-limiting value with increasing nbp. This decrease arises from the fluctuation in Wr and its neglect leads to an underestimate of C by 7 to 10%, even for short DNA with nbp approximately equal to 200. From an analysis of the experimental data of the two groups, the estimates of C = 3.1 to 3.2 X 10(-19) erg cm and lambda-1 = 1000 to 1200 A are obtained.     

Superhelical DNA with local substructures. A generalization of the topological constraint in terms of the intersection number and the ladder-like correspondence surface

The presence of certain local structural elements in superhelical DNA, such as cruciforms and denatured loops, complicates the topological and geometric analysis of these molecules. In particular, the duplex axis is often difficult to define. In consequence, the usual conservation condition, Lk = Tw + Wr, is often inapplicable as formulated in terms of the winding of either strand of the DNA about the duplex axis. We present here a more general formulation of the topological conservation condition in terms of a model in which the two strands of DNA are regarded as twisting about one another, and in which one of the two strands is considered to writhe. We define a ladder-like correspondence surface, which connects the two strands nd is independent of whether or not a unique duplex axis is locally available. These considerations lead to the definition of a new topological property of superhelical DNA, the intersection number, In. This quantity describes the complexity of a local structural element; in the case of a cruciform, for example, the intersection number is a measure of the number of duplex turns removed from the major segment of the DNA by the cruciform formation. In more general terms, the topological constraint applicable to closed circular DNA is given by Lk(W,C) + In(S,C) = Tw(W,C) + Wr (C), where W and C represent the two strands of the DNA and S is the ladder-like correspondence surface that connects the two strands.     

DNA topology in chromosomes: a quantitative survey and its physiological implications

Using a simple geometric model, we propose a general method for computing the linking number of the DNA embedded in chromatin fibers. The relevance of the method is reviewed through the single molecule experiments that have been performed in vitro with magnetic tweezers. We compute the linking number of the DNA in the manifold conformational states of the nucleosome which have been evidenced in these experiments and discuss the functional dynamics of chromosomes in the light of these manifold states.     

Theory of Site-Specific DNA-Protein Interactions in the Presence of Conformational Fluctuations of DNA Binding Domains

We develop a theory that explains how the thermally driven conformational fluctuations in the DNA binding domains (DBDs) of the DNA binding proteins (DBPs) are effectively coupled to the one-dimensional searching dynamics of DBPs for their cognate sites on DNA. We show that the rate γ_opt, associated with the flipping of conformational states of DBDs beyond which the maximum search efficiency of DBPs is achieved, varies with the one-dimensional sliding length L as γ_opt ∝ L⁻² and with the number of roadblock protein molecules present on the same DNA m as γ_opt ∝ m². The required free energy barrier E_RTO associated with this flipping transition seems to be varying with L as E_RTO ∝ ln L². When the barrier height associated with the conformational flipping of DBDs is comparable with that of the thermal free energy, then the possible value of L under in vivo conditions seems to be L ≤ 70 bps.     

Environmental influences on DNA superhelicity. The effect of ionic strength on superhelix conformation in solution

The techniques of small-angle X-ray scattering and analysis that have been developed by the authors are used to investigate the influence of ionic strength on the superhelical conformation of native COP608 plasmid DNA in solution. For salt concentrations below 0.1 M, the superhelicity is partitioned between twisting (Tw) and writhing (Wr) in the ratio delta Tw/Wr = 2. Near the physiological salt concentration, [Na+] = 0.2 M, a co-operative transition is observed in which the pitch angle of the toroidal superhelix is drastically decreased. This results in an almost complete relaxation of writhe. At salt concentrations in excess of the threshold for this transition, the superhelical partitioning occurs in the ratio delta Tw/Wr greater than 25. Energetic considerations support the suggestion that this transition results from co-operative, superhelical B to Z transconformation reactions at susceptible sites. A method is discussed that will enable the direct measurement of this secondary structural transition by means of X-ray scattering.     

Variance of Writhe for Wormlike DNA Rings with Excluded Volume

Abstract

We have calculated the variance of the equilibrium distribution of a circular wormlike polymer chain over the writhing number, <((Wr)² )>, with allowance for the excluded volume effects. Within this model the <((Wr)² )> value is a function of the number of Kuhn statistical segments, n, and the chain diameter, d measured in Kuhn statistical lengths, b. Simulated DNA chains varied from 200 to 10,000 base pairs and the d value varied from 0.02 to 0.2. Theory predicts a considerable ionic strength dependence of the DNA superhelix energy as a consequence of the change in the DNA diameter. A comparison with the available experimental data has yielded an estimate of the DNA torsional rigidity, the Kuhn statistical length, and the effective diameter of the double helix under conditions of the complete screening of the DNA electrostatic potential.     

Copy Number Variation of CCL3-like Genes Affects Rate of Progression to Simian-AIDS in Rhesus Macaques (Macaca mulatta)

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10⁻⁶) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se.     

Evidence for the presence of ribonucleotides at the 5' termini of some DNA molecules isolated from Escherichia coli polAex2.

We have purified a set of small DNA molecules from various strains of exponentially growing Escherichia coli, including E. coli polAex2. This material included very short molecules (2 S), the nascent DNA (“Okazaki fragments”) and some longer molecules. Most of the [³H]thymidine incorporated during a brief period of labeling was found in the 5 S to 15 S Okazaki fragments. There was a large number of the 2 S molecules in the cell. The properties of the 5′ ends of these molecules were investigated using three procedures. (1) The DNA preparation, pulse-labeled with [³H]thymidine, was reacted with polynucleotide kinase and ATP to insure that all 5′ ends were phosphorylated. After subjection of the DNA to alkaline hydrolysis, the proportion of incorporated ³H pulse-label that became susceptible to digestion by spleen exonuclease was determined. In different experiments there was an increment of up to 20% in the amount of pulse-labeled E. coli polAex2 DNA that could be hydrolyzed by the exonuclease after treatment with alkali. (2) As in the preceding protocol, phosphorylation of the 5′ ends was assured by reaction with kinase and ATP; the preparation was then treated with alkali and the number of 5′-OH ends generated that could be labeled with ³²P using [γ-³²P]ATP and kinase in a second reaction was determined. The data indicated that 3 to 30% of the molecules could be labeled after alkali digestion, but not before. (3) The DNA molecules were reacted with kinase and [γ-³²P]ATP after having been exposed previously to alkaline phosphatase. The end-labeled molecules were then subjected to an alkaline hydrolysis and the resulting hydrolysate chromatographed on a polyethyleneimine-cellulose thinlayer plate. Alkali treatment was found to release 2′(3′),5′-ribonucleoside diphosphates from 1 to 30% of the molecules; pAp and pGp predominated. Control experiments showed that these ribonucleotides were covalently linked to the 5′ ends of polydeoxyribonucleotides. Curiously, the smaller the DNA molecule the less likely it was to possess a 5′-terminal ribonucleotide. Very few apparent RNA/DNA molecules were observed in the non-polAex2 strains tested. These observations are in part in agreement with previous reports, and we infer that at least some of the nascent E. coli polAex2 DNA molecules are initiated in vivo with a ribonucleotide primer. The relatively smaller proportion of molecules with apparent 5′-terminal ribonucleotides among the smaller DNA molecules and in strains other than E. coli polAex2 suggests to us that there may exist a mechanism for initiating DNA molecules that does not require an RNA primer.     

A theory of thermal fluctuations in DNA miniplasmids.

A recent analysis of the normal modes of vibration of a ring formed by bringing together and sealing, with or without the addition of twist, the ends of rods that are straight when stress free is taken as the basis for a theory of the statistical thermodynamics of a canonical ensemble of DNA minicircles with specified linking number difference deltaLk and number N of base pairs. It is assumed that N corresponds to a circumference in the range of one or two persistence lengths. For such an ensemble, the theory yields an expression for the average writhe (Wr), which can be employed to calculate the free energy, entropy, and enthalpy of supercoiling, deltaGsc, deltaSsc, and deltaHsc. The results obtained for the dependence of deltaGsc on deltaLk and N are in accord with experimental observations of equilibrium distributions of topoisomers of plasmids with N approximately 200 bp.     

Topological distributions and the torsional rigidity of DNA. A Monte Carlo study of DNA circles

Distributions of the linking number of circular DNA molecules, defined as the sum of twist and the writhing number, are obtained by Monte Carlo simulations of small, randomly closed DNA circles. We estimate the relative contributions of fluctuations in twist and writhe to the linking number distribution, as functions of DNA size. Published experimental data on topoisomer distributions in circular DNA molecules are interpreted to estimate the torsional rigidity of DNA in solution. We show that ignoring the writhe component of the linking number distribution, even for DNA circles as small as 250 base-pairs, leads to an underestimate for the torsional stiffness of the double helix. The value of the torsional modulus obtained from this analysis, C = 3.4 X 10(-19) erg cm, is from 10 to 40% larger than that estimated by others and more than twice as large as the values obtained from fluorescence depolarization or other time-resolved spectroscopic measurements. We also develop further the theoretical treatment of ring closure probabilities for DNA described in the previous article. It is shown that the torsional part of the ring closure probability, phi 0,1 (tau 0) is a periodic function of DNA length that contributes strongly to the ring closure probability for short chains but makes negligible contributions for chains over 1000 base-pairs in length.     

Cloning and expression in Escherichia coli of Clostridium thermocellum DNA encoding β-glucosidase activity

Sau3A fragments of Clostridium thermocellum (NCIB 10682) DNA were ligated into the BamHI site of pBR322 and expressed in a Lac⁻mutant of Escherichia coli HB101. Five clones expressing β-glucosidase activity were shown by restriction enzyme analysis to contain a common 4.4 kbp fragment of inserted DNA. Hybridization of recombinant plasmids with chromosomal DNA ratified the physical maps of the inserted DNA and was further used to confirm that the 4.4 kbp fragment was common to all five clones. Enzyme activity, comprising cellobiase and aryl-β-glucosidase, was similar with respect to substrate specificity for each of the five clones, and was expressed independently of the orientation of the cloned DNA. A differential effect of temperature on activity of the cellobiase and aryl-β-glucosidase activities was observed but in other respects, the properties of the cloned β-glucosidase corresponded to those of the single β-glucosidase previously described for C. thermocellum.     

Action at Hooked or Twisted-Hooked DNA Juxtapositions Rationalizes Unlinking Preference of Type-2 Topoisomerases

The mathematical basis of the hypothesis that type-2 topoisomerases recognize and act at specific DNA juxtapositions has been investigated by coarse-grained lattice polymer models, showing that selective segment passages at hooked juxtapositions can result in dramatic reductions in catenane and knot populations. The lattice modeling approach is here extended to account for the narrowing of variance of linking number (Lk) of DNA circles by type-2 topoisomerases. In general, the steady-state variance of Lk resulting from selective segment passages at a specific juxtaposition geometry j is inversely proportional to the average linking number, 〈Lk〉_j, of circles with the given juxtaposition. Based on this formulation, we demonstrate that selective segment passages at hooked juxtapositions reduce the variance of Lk. The dependence of this effect on model DNA circle size is remarkably similar to that observed experimentally for type-2 topoisomerases, which appear to be less capable in narrowing Lk variance for small DNA circles than for larger DNA circles. This behavior is rationalized by a substantial cancellation of writhe in small circles with hook-like juxtapositions. During our simulations, we uncovered a twisted variation of the hooked juxtaposition that has an even more dramatic effect on Lk variance narrowing than the hooked juxtaposition. For an extended set of juxtapositions, we detected a significant correlation between the Lk narrowing potential and the logarithmic decatenating and unknotting potentials for a given juxtaposition, a trend reminiscent of scaling relations observed with experimental measurements on type-2 topoisomerases from a variety of organisms. The consistent agreement between theory and experiment argues for type-2 topoisomerase action at hooked or twisted-hooked DNA juxtapositions.