Replication of herpesvirus DNA. III. Rate of DNA elongation.

The rate of pseudorabies virus DNA elongation was measured by three different techniques: density shift experiments, radioautography examined by light microscopy, and radioautography examined by electron microscopy. The rate of the fork movement at 37 degrees C was estimated to be approximately 1 micron/min.     

Adenovirus type 2 DNA replication. II. Termini of DNA replication.

Complete, mature adenovirus type 2 DNA molecules were isolated from virus-infected HeLa cells, pulse-labeled at 20 h postinfection in [3H]thymidine pulses shorter than the time necessary for one round of viral DNA replication. After digestion with the restriction endonucleases Eco RI, Hpa I, and Hind III, a temporal order of synthesis of different regions of the viral genome was established from the relative specific radioactivities in the restriction enzyme fragments. A comparison with the physical order of these fragments revealed the existence of two termini of DNA replication towards both the molecular right and left ends, respectively, of the viral chromosome.     

Site-specific cleavage of DNA by E. coli DNA gyrase.

E. coli DNA gyrase, which catalyzes the supercoiling of DNA, cleaves DNA site-specifically when oxolinic acid and sodium dodecylsulfate are added to the reaction. We studied the structure of the gyrasecleaved DNA because of its implications for the reaction mechanism and biological role of gyrase. Gyrase made a staggered cut, creating DNA termini with a free 3' hydroxyl and a 5' extension that provided a template primer for DNA polymerase. The cleaved DNA was resistant to labeling with T4 polynucleotide kinase even after treatment with proteinase K. Thus the denatured enzyme that remains attached to cleaved DNA is covalently bonded to both 5' terminal extensions. The 5' extensions of many gyrase cleavage fragments from phi X174, SV40 and Col E1 DNA were partially sequenced using repair with E. coli DNA polymerase I. No unique sequence existed within the cohesive ends, but G was the predominant first base incorporated by DNA polymerase I. The cohesive and sequences of four gyrase sites were determined, and they demonstrated a four base 5' extension. The dinucleotide TG, straddling the gyrase cut on one DNA strand, provided the only common bases within a 100 bp region surrounding the cleavage sites. Analysis of other cleavage fragments showed that cutting between a TG doublet is common to most, or all, gyrase cleavages. Other bases common to some of the sequenced sites were clustered nonrandomly around the TG doublet, and may be variable components of the cleavage sequence. This diverse recognition sequence with common elements is a pattern shared with several other specific nucleic acid-protein interactions.     

Rat hepatoma cells nucleolar DNA. 2. Analysis of nucleolar DNA.

The analysis by CsCl density gradient of nucleolar DNA has revealed that the 1.700 g/cm3 main component can be subdivided in three subcomponents with buoyant densities of 1.707 g/cm3, 1.700 g/cm3, 1.690 g/cm3. The 1.707 g/cm3 and 1.690 g/cm3 components contain all the repetitive sequences which comprise 15 % of the total nucleolar DNA. The ribosomal cistrons are found in components having buoyant density between 1.707 g/cm3 and 1.725 g/gm3. Sodium-p-aminosalicylate-DNA interactions have revealed that only the 1.700 g/cm3 fraction has a destabilized secondary structure. The possible localization of these different fractions on peri and intranucleolar fractions is discussed.     

Synthesis of non-natural DNA using DNA polymerase.

Nonnatural nucleotide modified by glucose or galactose was synthesized to increase functional diversity of DNA library. These compounds were incorporated in a DNA double strand using Klenow Fragment as well as dTTP. These functional group could be ordered sequentially on a DNA double strand at intervals of few angstroms according to the designed template sequence within a few hours. This method must be useful to constructing nonnatural DNA library or designed supramolecular structures.     

Wrinkled DNA.

The B form of poly d(GC):poly d(GC) in orthorhombic microcrystallites in oriented fibers has a secondary structure in which a dinucleotide is the repeated motif rather than a mononucleotide as in standard, smooth B DNA. One set of nucleotides (probably GpC) has the same conformations as the smooth form but the alternate (CpG) nucleotides have a different conformation at C3'-O3'. This leads to a distinctive change in the orientation of the phosphate groups. Similar perturbations can be detected in other poly d(PuPy):poly d(PuPy) DNAs such as poly d(IC):poly d(IC) and poly d(AT):poly d(AT) in their D forms which have tetragonal crystal environments. This suggests that such perturbations are intrinsic to all stretches of duplex DNA where purines and pyrimidines alternate and may play a role in the detection and exploitation of such sequences by regulatory proteins.     

Heteronomous DNA.

A fibrous form of poly d(A):poly d(T) has a heteronomous secondary structure which is the first to be confirmed for a polynucleotide duplex: although both chains are 10(1) helices, mutually hydrogen-bonded in the standard (Watson-Crick) fashion, each has a quite different conformation. One chain -- probably poly d(A) -- has C3'-endo-puckered furanose rings characteristic of the A family of polynucleotide secondary structures while the other -- probably poly d(T) -- has the C2'-endo-puckered rings of the B family. Since analogous heteronomous structures could be assumed by DNA-DNA or DNA-RNA duplexes containing more general base sequences the polymorphic range of polynucleotide double-helices may be even greater than we have come to suppose.     

DNA topoisomerases.

DNA topoisomerases play an important role in regulating DNA structure, thus affecting many aspects of chromosome function inside cells. Recent progress in this field raises exciting questions regarding the distinct and critical functions of multiple topoisomerases, and the roles of DNA topoisomerases in the processes of chromosome condensation, decondensation, and segregation.     

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.     

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.     

DNA deoxyribophosphodiesterase.

A previously unrecognized enzyme acting on damaged termini in DNA is present in Escherichia coli. The enzyme catalyses the hydrolytic release of 2-deoxyribose-5-phosphate from single-strand interruptions in DNA with a base-free residue on the 5' side. The partly purified protein appears to be free from endonuclease activity for apurinic/apyrimidinic sites, exonuclease activity and DNA 5'-phosphatase activity. The enzyme has a mol. wt of approximately 50,000-55,000 and has been termed DNA deoxyribophosphodiesterase (dRpase). The protein presumably is active in DNA excision repair to remove a sugar-phosphate residue from an endonucleolytically incised apurinic/apyrimidinic site, prior to gap filling and ligation.     

Interaction of DNA with DNA binding proteins. III. Infectivity of protein-complexed phage fd DNA in Escherichia coli spheroplasts.

Complex formation of circular, single-stranded phage fd DNA with Escherichia coli DNA binding protein HD or phage fd gene 5 protein keeps infection of E. coli spheroplasts at the level of free phage DNA, whereas complexes of this DNA with E. coli DNA unwinding protein show a strongly reduced efficiency of transfection. Displacement of the unwinding protein by HD protein or gene 5 protein also maintains the poor adsorption of the complexes to spheroplasts. Free E. coli DNA unwinding protein and residual amounts of this protein bound to the DNA may interfere with the adsorption and the uptake of the phage genome.     

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.