Application of hypoxia-induced shut down of replicon initiation to the analysis of replication intermediates in Ehrlich ascites cells

Cultured Ehrlich ascites cells were subjected to hypoxic conditions for about 2 h, then reaerated or allowed to remain hypoxic. The newly formed DNA of hypoxic or reaerated cells was labeled with [3H]thymidine using different pulse and pulse/pulse-chase protocols. The chain length distribution of the labeled DNA molecules was analysed by sedimentation after lysing the cells on the top of alkaline sucrose gradients. The results indicated that the hypoxia effectively and reversibly suppressed the initiation of new replication units. Initiation, growth and integration of Okazaki pieces into active replicons was not noticeably affected. In marked contrast to aerobic cells, the use of hypoxic cells allows the separation of Okazaki pieces as a distinct class of pulse labeled short DNA chains. Short daughter DNA of very recently initiated replicons did not interfere at pulse times shorter than 4 min. For examination of the newly initiated replicons it seems favourable to trigger a burst of initiations by reaeration.     

The rate of DNA replication in the polytene chromosomes of Rhynchosciara angelae

The distribution pattern of 5-bromodeoxyuridine-labelled DNA from salivary glands of Rhynchosciara angelae upon caesium chloride gradient centrifugation was studied with DNA of different molecular weights. This pattern suggests a very low rate of DNA chain growth in polytene chromosomes. The rate of DNA chain growth was found to be 0.025 μm/min at 24 °C. The result was obtained through the development of a mathematical expression which took into account the distribution of the 5-bromodeoxyuridine-labelled DNA in CsCl gradients.DNA pulse-labelled for a short time sediments more slowly in alkaline sucrose gradients than DNA which has been labelled during a prolonged incubation. However, in neutral sucrose gradients the pattern of banding is the same for both DNAs. This indicates a discontinuity in the newly synthesized DNA strand, but not in the template strand. The transition of slow sedimenting to fast sedimenting DNA observed in alkaline sucrose gradients, occurs very slowly, as would be expected for a slow rate of DNA chain growth.The data obtained provided a means of comparing the number of replication points with the rate of DNA chain elongation and the length of S phase in Rhynchosciara.     

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.     

RNA primers in Simian virus 40 DNA replication. II. Distribution of 5' terminal oligoribonucleotides in nascent DNA.

A cell-free simian virus 40 (SV40) DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages were found to copurify with SV40 replicating DNA. These linkages were identified by transfer of a fraction of the ³²P from the 5′ position of a deoxyribonucleotide to 2′(3′)rNMPs upon either alkaline hydrolysis or RNAase T₂ digestion of SV40 replicating [³²P]DNA. Alkaline hydrolysis also exposed 5′ terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with P1 nuclease. The RNA-DNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5′ termini (RNA primers). The density of a portion of the Okazaki pieces in potassium iodide gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a defined length in the presence of either RNAase T₂ or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least seven-residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that one-third to one-half of them contained rN-³²P-dN linkages, the oligoribonucleotides must be covalently attached to the 5′ ends of nascent DNA chains. Although the significance of two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers.Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in nascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5′ end of growing daughter strands.³²P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII + III restriction fragments of SV40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences.     

Single-stranded regions in DNA isolated from different developmental stages of the sea urchin

Long-term labeled sea urchin embryo (Strongylocentrotus purpuratus) DNAs were examined for size of recovered pieces, single-strandedness, and length of continuous double-stranded regions. Sizing on neutral sucrose gradients indicates that morula stage DNA sediments predominantly at 31 S, blastula stage DNA at 27 S, and gastrula stage DNA as a broad range of sizes of greater than 29 S. Treatment of [3H]thymidine-labeled DNA with Aspergillus oryzae S1 nuclease removes 19% of the 3H from morula stage DNA, 4% of the 3H from blastula stage DNA, and less than 0.1% of the 3H from gastrula stage DNA. Sedimentation of S1 nuclease treated [3H]DNAs on alkaline sucrose gradients indicates that in native morula stage DNA there is a nick or gap in one strand approximately every 9700 base pairs, in native blastula stage DNA about every 3300 base pairs, and very few nicks or gaps in native gastrula stage DNA.     

Alkali lability of bacteriophage phi W-14 DNA.

The molecular weight of bacteriophage phi W-14 DNA, determined by velocity sedimentation in neutral sucrose gradients, was 92 +/- 6 X 10(6). The DNA showed marked fragmentation in alkaline sucrose gradients. This fragmentation was not a consequence of preexisting single-strand interruptions in the DNA, since thermal denaturation of DNA yielded intact single strands. The alpha-putrescinylthymine groups in phi W-14 DNA appeared to be labile; some, or parts of some, of these groups were cleaved from the DNA in alkali.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

Sedimentation velocity of DNA in isokinetic sucrose gradients: calibration against molecular weight using fragments of defined length.

The relationship between sedimentation coefficient and molecular weight for DNA sedimenting in preformed alkaline and neutral sucrose gradients was determined using absolute molecular weight standards (restriction fragments of plasmid pBR322 and phage lambda DNA). The range of calibration for alkaline gradients was extended to small DNA fragments (652 base-pairs) for the first time. The exponent b in the equation S20 degrees, w = aMb was found to be 0.380 in neutral gradients and 0.410 in alkali. The latter value differs significantly from previous estimates. The gradients were isokinetic, and the distance sedimented was shown to be directly proportional to the sedimentation coefficient at all times.     

Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.     

Chemical and physical properties of adeno-associated satellite virus DNA produced during coinfection with herpes simplex virus

Infectious DNA from adeno-associated satellite virus (ASV) has been isolated from cells coinfected with a temperature-sensitive mutant of herpes simplex virus (HSV) type 1 in the absence of contaminating HSV DNA. This satellite virus DNA does not appear to differ in its physical, chemical and biological properties from DNA isolated directly from virions or from cells co-infected with adenovirus. The DNA is double-stranded with a buoyant density of 1.718 gm/cm³. It sediments at 16S in both neutral and alkaline sucrose gradients. Single-stranded DNA from alkaline sucrose gradients has a modal length of 1.5 μm and demonstrates evidence of internal redundancies in the electron microscope.     

The mechanism of replication of phi X 174. XVIII. Gene A and A* proteins of phi X 174 bind tightly to phi X 174 replicative form DNA

Evidence is presented that the gene A and A * proteins of bacteriophage phi X 174 form covalent associations with the 5' ends of the DNA molecules when superhelical phi X replicative form DNA is nicked by a combination of these proteins in vitro. This evidence is: 1, The 5' ends of the DNA molecules nicked by the gene A protein and reacted with bacterial alkaline phosphatase were protected against subsequent phosphorylation by polynucleotide kinase even after treatment of the nicked DNA with SDS and pronase followed by centrifugation on a high-salt neutral sucrose gradient. 2, Iodinated pronase-sensitive material remained attached to the nicked replicative form DNA and could not be removed by exposure to SDS or 2 M NaCl, either by sedimentation through high-salt neutral sucrose gradients, or by CsCl equilibrium centrifugation. 3, Iodinated pronase-sensitive material was detected on DNA that had been nicked during the reaction, but not on unreacted DNA. 4, Electrophoresis of the iodinated pronase-sensitive, DNA-bound material in SDS-polyacrylamide gels after DNAse digestion revealed that it was composed almost entirely polypeptides with electrophoretic mobilities similar to those of the gene A and A * proteins. We speculate that the gene * protein may be essential for normal progeny single-stranded DNA synthesis in vivo.     

Factors influencing the ability of isolated cell nuclei to from gels in dilute alkali

1. Known methods for isolating cell nuclei are divided into two classes, depending on whether or not the nuclei are capable of forming gels in dilute alkali or strong saline solutions. Methods which produce nuclei that can form gels apparently prevent the action of an intramitochondrial enzyme capable of destroying the gel-forming capacity of the nuclei. Methods in the other class are believed to permit this enzyme to act on the nuclei during the isolation procedure, causing detachment of DNA from some nuclear constituent (probably protein). 2. It is shown that heating in alkaline solution and x-irradiation can destroy nuclear gels. Heating in acid or neutral solutions can destroy the capacity of isolated nuclei to form gels. 3. Chemical and biological evidence is summarized in favor of the hypothesis that DNA is normally bound firmly to some nuclear component by non-ionic linkages.     

FACTORS INFLUENCING THE ABILITY OF ISOLATED CELL NUCLEI TO FORM GELS IN DILUTE ALKALI

1. Known methods for isolating cell nuclei are divided into two classes, depending on whether or not the nuclei are capable of forming gels in dilute alkali or strong saline solutions. Methods which produce nuclei that can form gels apparently prevent the action of an intramitochondrial enzyme capable of destroying the gel-forming capacity of the nuclei. Methods in the other class are believed to permit this enzyme to act on the nuclei during the isolation procedure, causing detachment of DNA from some nuclear constituent (probably protein). 2. It is shown that heating in alkaline solution and x-irradiation can destroy nuclear gels. Heating in acid or neutral solutions can destroy the capacity of isolated nuclei to form gels. 3. Chemical and biological evidence is summarized in favor of the hypothesis that DNA is normally bound firmly to some nuclear component by non-ionic linkages.     

Size and Composition of Marek''s Disease Virus Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from purified nucleocapsids of Marek's disease herpesvirus (MDV) was cosedimented with T4 and with herpes simplex virus (HSV) DNA in neutral sucrose density gradients and with T4 DNA in alkaline sucrose density gradients. These experiments indicated that the intact MDV DNA had a sedimentation constant of 56S corresponding to a molecular weight of 1.2 x 10(8) daltons. In the alkaline gradients, the largest and most prominent band contains a DNA sedimenting at 70S corresponding to 6.0 x 10(7) daltons in molecular weight. The DNA is therefore double-stranded and not cross-linked. Isopycnic sedimentation of the MDV DNA molecules with SPO1, Micrococcus lysodeikticus, and HSV DNA gave a density of 1.705 g/cm(3) corresponding to 46 guanine plus cytosine moles per cent. Lastly, in hybridization tests the DNA hybridized with RNA of infected cells but not with that of uninfected cells supporting the conclusion that it is viral.     

Ribonucleotides in closed circular mitochondrial DNA from HeLa cells

Closed circular mitochondrial DNA from HeLa cells is sensitive to both alkali and ribonucleases. The kinetics of ring opening in alkali suggest at least two classes of molecules. One class undergoes rapid breakdown, ultimately to fragments smaller than unit length, in contrast to the second class, which is more resistant to alkaline cleavage and is converted in large part to unit length single strands. Ribonucleases A, T₁ and H relax the supercoiled molecules, indicating that the alkali susceptibility is due to the presence of ribonucleotides in the DNA. By comparison with the rate of hydrolysis of RNA, the alkali-resistant class of mitochondrial DNA molecules is estimated to contain approximately 3 ribonucleotides and the alkalisensitive class 10–18.     

The effect of thymine starvation on chromosomal structure of Escherichia coli JG-151

Labelled DNA extracted from control and thymine starved cells was qualitatively characterized with respect to sedimentation properties in alkaline sucrose gradients. DNA isolated from cells undergoing loss of division ability demonstrated decreasing sedimentation velocity. Sedimentation profiles of DNA extracted from cells which were starved for thymine under conditions which allowed spontaneous recovery of division ability to occur, demonstrated an increase in DNA sedimentation velocity toward normal control value. It appears that while thymine starvation can result in single strand breaks, this damage is not irreversible, for under certain conditions rejoining of the breaks can occur.     

The origin of nascent single-stranded DNA extracted from mammalian cells.

In vitro cultured bovine liver cells were labelled with radioactive thymidine and dissolved in 0.5% sodium dodecyl sulphate. Centrifugation of the lysate through sucrose gradients in a zonal rotor revealed a slowly sedimenting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CsCl density gradients, and sedimentation in neutral and alkaline sucrose gradients. It contained besides small amounts of fragmented bulk DNA, single-stranded nascent DNA and single-stranded pre-labelled DNA which could be separated from each other by using BrdU as a density label. The density labelling also revealed small amounts of nascent-nascent DNA duplexes. The slowly sedimenting fraction was practically absent from cell lysates which were prepared in 2 M NaCl - 50 microgram/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating DNA by branch migration. Pre-labelled single strands may be released by the same branch migration. Pre-labelled single strands may be released by the same mechanism, but the in vivo structure from which they originate has yet to be elucidated.     

DNA strand scission by the antitumor protein neocarzinostatin

The antibiotic protein, neocarzinostatin, induces the scission of DNA strands $\text{in vivo}$ and $\text{in vitro}$. HeLa cell DNA prelabelled with [¹⁴C] thymidine is cut into large pieces with a peak at 80–90S when cells are incubated with 0.5 to 5.0 μg/ml of highly purified neocarzinostatin. Incubation of the antibiotic (0.5 μg/ml) with [³H] SV40 DNA in the presence of 2-mercaptoethanol results in the conversion of superhelical DNA I to nicked circular duplex DNA II. At high levels of drug, smaller fragments of linear DNA are produced. Strand breaks are detected in both neutral and alkaline sucrose gradients, indicating that drug susceptibility is not due to alkali-labile bonds.     

Discontinuous DNA replication in mouse P-815 cells.

The length of newly synthesized DNA strands from mouse P-815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3-H]-Thymidine pulses of 2-8 min at 37 degrees C predominantly label molecules of 20-60 S. With 30-s pulses at 25 degrees C, all the [3-H]thymidine appears in short DNA strands of 50-200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5' end and the 3' end. In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single-stranded molecules. About 90% are resistant to hydrolysis by the single-strand-specific nuclease S-1 and band in isopycnic gradients at the buoyant density of double-stranded DNA. No evidence for ribonucleotides at the 5' end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [gamma-32P]ATP.     

Okazaki pieces grow opposite to the replication fork direction during simian virus 40 DNA replication.

Simian virus 40 replicating DNA was pulse labeled with alpha-32P-dATP using an acellular DNA replication system. Nascent DNA chains of less than 200 nucleotides (Okazaki pieces) were then isolated from the denatured replicating DNA by electrosieving through a polyacrylamide gel column. The purified Okazaki pieces were hybridized to separated strands of Bg1(1)+Hpa1 simian virus 40 DNA restriction fragments immobilized on nitrocellulose filters. Only strands with polarity of the DNA replication fork direction hybridized with Okazaki pieces. Hence, Okazaki pieces in simian virus 40 are synthesized against the DNA replication fork direction.