DNA supercoiling and relaxation by ATP-dependent DNA topoisomerases.

Bacterial DNA gyrase and the eukaryotic type II DNA topoisomerases are ATPases that catalyse the introduction or removal of DNA supercoils and the formation and resolution of DNA knots and catenanes. Gyrase is unique in using ATP to drive the energetically unfavourable negative supercoiling of DNA, an example of mechanochemical coupling: in contrast, eukaryotic topoisomerase II relaxes DNA in an ATP-requiring reaction. In each case, the enzyme-DNA complex acts as a 'gate' mediating the passage of a DNA segment through a transient enzyme-bridged double-strand DNA break. We are using a variety of genetic and enzymic approaches to probe the nature of these complexes and their mechanism of action. Recent studies will be described focusing on the role of DNA wrapping on the A2B2 gyrase complex, subunit activities uncovered by using ATP analogues and the coumarin and quinolone inhibitors, and the identification and functions of discrete subunit domains. Homology between gyrase subunits and the A2 homodimer of eukaryotic topo II suggests functional conservation between these proteins. The role of ATP hydrolysis by these topoisomerases will be discussed in regard to other energy coupling systems.     

DNA sequencing and helix-coil transition. I. Theory of DNA melting

An explicit analytic formula accurately describing the melting of a natural DNA is derived. For phage ?X-174 and virus SV-40, the nucleotide sequences of which are known, the formula fits experimental data for the differential melting curve almost within the experimental accuracy.     

DNA binding and antigenic specifications of DNA gyrase.

Complexes of DNA gyrase and minichromosomal DNA containing the origin of replication of Escherichia coli (oriC) can be formed without metabolic energy and visualised by electron microscopy. The A subunit, part of the A2B2-DNA gyrase complex is the binding protein. Various binding sites are scattered around the minichromosomal DNA including oriC. The minimal origin contains the only prominent and reproducible binding site. Binding to this site is suppressed by oxolinic acid and the ATP analogue beta-y-imido ATP. If gyrase isolated from the gram-positive bacterium Bacillus subtilis is used no binding to oriC is seen. This observation is consistent with antigenic differences between the A subunits of the two microorganisms. The binding to oriC might reflect a requirement for DNA gyrase during the initiation of DNA replication.     

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Summary Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 ) were present; instead, smaller circles characteristic for each mutant sudied were found. Eco Rl digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type.The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 . These circles ranged in size from 0.89 to greater than 20 ; only one molecule out of some 200 molecules was thought to be of full length (31 ). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm³) and a second at 1.699 g/cm³. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23×10⁶ daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20×10⁶ daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length.These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho ^- mutation in the yeast, S. cerevisiae.     

Thermodynamic and structural properties of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of identical sequence

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.     

Rifampin and vaccinia DNA.

The effect of rifampin on the replication of vaccinia DNA was studied in mouse L cells by a cytochemical techinque and by alkaline sucrose sedimentation analysis of newly synthesized viral DNA molecules. By the use of a fluorescent DNA-binding compound (Hoechst 33258), the sequential appearance, size, and location of the viral "factories" in rifampin-treated, virus-infected cells were found to be indistinguishable from those observed in untreated, infected cells. Sedimentation analysis in alkaline scurose gradients of the viral DNA molecules labeled in pulse-chase experiments showed that formation of small fragments, elongation into "intermediate"-sized molecules, and maturation into full-length viral DNA and, finally, into cross-linked viral DNA molecules occurred in the absence or presence of rifampin. The results support the view that the primary effect of the drug is related to assembly or morphogenesis.     

Denaturation and renaturation of DNA. I. Equilibrium statistics of copolymeric DNA

The one-dimensional Ising model, with nearest neighbor correlation only, suitably modified, is used to explain the observed linear dependence of melting temperature of copolymeric DNA with GC content. Transition curves are plotted for regular, random, and Markoff distribution of base pairs for various values of a correlation parameter U between nearest neighbor bonds. Exact analytic formulas are given for fraction of bonds intact at a particular temperature for various regular distributions for all U and approximate ones for random and Markoff distributions for small U. A scheme is indicated for further improvement. The model, in principle, makes it possible to estimate the statistical distribution of base pairs from the detailed shape of the transition curve.     

DNA sequencing and helix–coil transition. III. DNA sequencing

We show that the fine oscillatory structure of the DNA melting curve can be used to determine explicitly the nucleotide composition and the order of certain domains within the DNA. If DNA is specifically fragmented, the order of fragments can be learned directly from a comparison of the differential melting curves of the nonfragmented and fragmented DNA. The indicated information may complement exact methods of DNA sequencing. The proposed analysis is applied to bacteriophage ?X-174, whose melting curve is known. Compared to the known ?X-174 DNA sequence, the results of the analysis are found to be very accurate.     

Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.

DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.     

DNA methylation and development.

(1) Isolated rat liver mitochondria were subjected to catalytic hydrogenation using a water-soluble Pd complex and molecular H2. This treatment resulted in a reduction of double bonds on phospholipid acyl chains as judged by gas chromatography of fatty acid methyl esters and HPLC of dinitrobenzoyldiacylglycerols. (2) After hydrogenation, mitochondria lost their ability to hydrolyze endogenous phospholipids in alkaline, Ca2+ containing medium, while phospholipase A2 retained full activity against exogenous substrates, regardless of whether those substrates were hydrogenated or not. (3) Inhibition by hydrogenation of endogenous phospholipid hydrolysis correlated with the loss of polyunsaturated fatty acyls, rather than with changes of the bulk membrane fluidity as measured by ESR and fluorescence studies. (4) These data suggest that the unsaturation of mitochondrial membrane lipids might be important for regulation of phospholipid breakdown by endogenous phospholipases. In particular, polyunsaturated molecular species seem to be involved in making phospholipids accessible to phospholipase A-mediated hydrolysis.     

DNA probes and automation.

Current methods for DNA probe analysis in principle provide access to the total genetic information of any organism and permit applications in health care, for example. In practice, however, greatly improved efficiency of methods for decoding genetic information is required for both research and routine applications. This review describes recent progress towards automated DNA probe assays.     

Glucocorticoid receptor-steroid complex binding to DNA. Competition between DNA and DNA-cellulose.

The binding of the glucocorticoid receptor-steroid complex from a line of rat hepatoma tissue culture (HTC) cells to DNA has been examined. An equilibrium competition assay involving a constant, low total amount of double-stranded DNA was developed to compare the complex binding ability of DNA free in solution and bound to cellulose. This binding ability is lowered by a factor of five when DNA is associated with cellulose. Similar studies with HTC cell, calf-thymus, and Escherichia coli DNA revealed no difference in the relative number or affinity of binding sites for receptor-steroid complex in each DNA. The synthetic DNA molecules poly[d(A-T)-d(A-T)] and poly[d(G-C)-d(G-C)] bound complexes equally well but less than the three "natural" DNA molecules. This appears to be due to differences in acceptor site affinity and suggests that nucleotide complexity and/or sequence influences the affinity of HTC cell receptor-glucocorticoid complexes for DNA.