Synthesis of complex forms of bacteriophage phiX174 double-stranded DNA in a temperature-sensitive dnaC mutant of Escherichia coli C.

Fast-sedimenting forms of bacteriophage phiX174 double-stranded replicative-form DNA observed in normal infections continued to accumulate at the nonpermissive temperature in a temperature-sensitive dnaC mutant of Escherichia coli. These complex molecules accounted for up to half of the DNA synthesized during short pulses at the nonpermissive temperature. They were the dead-end products of DNA synthesis, not intermediates in normal replicative-form replication. The data suggest that these higher-than-normal-molecular-weight DNA molecules result from abnormal initiation of phiX174 replicative-form DNA replication.     

Temperature-sensitive deoxyribonucleic acid replication in a dnaC mutant of Bacillus subtilis.

A temperature-sensitive mutant of Bacillus subtilis is defective in deoxyribonucleic acid (DNA) synthesis, contains a lesion in the dnaC locus, and is not primarily an initiation mutant. The amount of DNA synthesized by this mutant at temperatures above 40 C decreases with increasing temperature. DNA synthesis resumes within 20 min after the temperature is lowered to 30 C. In the presence of chloramphenical, DNA synthesis begins at a reduced rate after the temperature is lowered to 30 C. Spores germinated at 46 C cannot initiate DNA replication. The capacity for residual DNA synthesis is stable at the restrictive temperature during inhibition of DNA synthesis. When the temperature is lowered to 30 C after a period of incubation at 43 C, DNA synthesis starts at the origin of the chromosome as well as at preexisting growing points. Similar DNA synthesis patterns are found in mutant cells in vivo and after toluene treatment.     

Regulation of DNA synthesis and capacity for initiation in DNA temperature mutants of Escherichia coli. III. Synthesis of the dnaA protein and of DNA-binding proteins

Summary The synthesis and action of the dnaA product with respect to DNA initiation and the synthesis of DNA-binding proteins in Escherichia coli was examined. Results indicate that when dnaA product is irreversibly denatured and must be synthesized before initiation can occur, its synthesis and action appear to be complete approximately 30 min before initiation takes place. However, in mutants whose dnaA product is temperature reversible the action of the dnaA product appears to occur near the time of initiation. Examination of the DNA-binding proteins from the mutants suggests that a 53 kd protein, possibly the dnaA product, may be synthesized at the time of initiation under normal conditions at permissive temperature. The presence of active dnaA product appears to trigger the synthesis of a 60–65 kd protein which may be responsible for preventing another immediate initiation event.     

Temperature-Sensitive Initiation of Chromosome Replication in a Mutant of Bacillus subtilis

A mutant of Bacillus subtilis Ts37 has been isolated in which deoxyribonucleic acid (DNA) synthesis is inhibited at high temperature. The results presented here indicate that the process of initiation of DNA replication is temperature sensitive in this mutant. After shifting to 45 C, DNA increases 40 to 50% before synthesis ceases; an inhibition of protein synthesis permits an equivalent amount of DNA to be synthesized. A density shift experiment coupled with a marker frequency analysis shows that DNA synthesized at 45 C is highly enriched in the markers situated at the end of the chromosome. Transforming DNA extracted from a culture which has been incubated at 45 C exhibits the relative transforming efficiency for origin and terminus markers characteristic of completed chromosomes. After a shift back from 45 C to 30 C, reinitiation appears to occur always in the same region of the bacterial chromosome; in addition, replication as well as cell division is synchronized.     

The dnaK gene of Escherichia coli functions in initiation of chromosome replication.

A newly isolated dnaK mutant of Escherichia coli, which contains the mutation dnaK111, has been found to be conditionally defective in initiation of DNA replication. Mutant cells that were transferred to high temperature exhibited residual DNA synthesis before the synthesis stopped completely. Analysis of the DNA synthesized at high temperature by hybridization with probe DNAs for detection of DNA replicated in the origin (oriC) and terminal (terC) regions has revealed that this mutant is unable to initiate a new round of DNA replication at high temperature after termination of the round in progress. The cells exposed to high temperature were subsequently capable of initiating DNA replication at low temperature in a synchronous manner. DNA synthesis of this mutant became temperature resistant upon inactivation of the rnh gene, similar to that of dnaA mutants, although cell growth of the dnaK mutant with the inactive rnh gene remained temperature sensitive. The dnaK mutation prevented DNA synthesis of lambda bacteriophage at high temperature even in the absence of the rnh gene function.     

Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutations in a 135,000-Mr subunit of the virion-associated DNA-dependent RNA polymerase

The phenotypic defects of three temperature-sensitive (ts) mutants of vaccinia virus, the ts mutations of which were mapped to the gene for one of the high-molecular-weight subunits of the virion-associated DNA-dependent RNA polymerase, were characterized. Because the virion RNA polymerase is required for the initiation of the viral replication cycle, it has been predicted that this type of mutant is defective in viral DNA replication and the synthesis of early viral proteins at the nonpermissive temperature. However, all three mutants synthesized both DNA and early proteins, and two of the three synthesized late proteins as well. RNA synthesis in vitro by permeabilized mutant virions was not more ts than that by the wild type. Furthermore, only one of three RNA polymerase activities that was partially purified from virions assembled at the permissive temperature displayed altered biochemical properties in vitro that could be correlated with its ts mutation: the ts13 activity had reduced specific activity, increased temperature sensitivity, and increased thermolability under a variety of preincubation conditions. Although the partially purified polymerase activity of a second mutant, ts72, was also more thermolabile than the wild-type activity, the thermolability was shown to be the result of a second mutation within the RNA polymerase gene. These results suggest that the defects in these mutants affect the assembly of newly synthesized polymerase subunits into active enzyme or the incorporation of RNA polymerase into maturing virions; once synthesized at the permissive temperature, the mutant polymerases are able to function in the initiation of subsequent rounds of infection at the nonpermissive temperature.     

Low-Temperature-Induced DnaA Protein Synthesis Does Not Change Initiation Mass in Escherichia coli K-12

Expression of the dnaA gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14 degrees C than at 37 degrees C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30 degrees C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of dnaA gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.     

Mechanism of mitochondrial DNA replication in mouse L-cells: kinetics of synthesis and turnover of the initiation sequence

Pulse-chase radioactive labeling experiments using thymidine kinase-plus mouse LA9 cells have shown that the 7 S mitochondrial DNA initiation sequence of mitochondrial DNA is synthesized and turned over at a faster rate than previously determined. These pulse-chase labeling experiments have also determined that the replication time of mouse LA9 cell mitochondrial DNA is one hour. The halflife of pulse-labeled 7 S mitochondrial DNA initiation sequences is approximately 70 minutes. This turnover is so rapid that at least 95% of the mitochondrial DNA initiation sequences synthesized are lost to turnover without acting as primers for expansion synthesis of the mitochondrial DNA heavy strand. The mechanism of 7 S mitochondrial DNA turnover does not lead to significant accumulation of free 7 S mitochondrial DNA single-strands within mitochondria. Resynthesis of the 7 S mitochondrial DNA initiation sequence is sufficiently rapid that the majority of mitochondrial DNA molecules are maintained as displacement loop molecules. Approximately 20% of all nucleotides polymerized into mitochondrial DNA are incorporated into the 7 S initiation sequences. The size of newly synthesized 7 S mitochondrial DNA strands varies from about 500 to 620 nucleotides. Several size classes are resolved by polyacrylamide/urea gel electrophoresis and each class has approximately the same turnover rate.Mouse LD cells maintain their mitochondrial DNA genomes as unicircular, head-to-tail dimers. Since a significant fraction of these unicircular dimers contain only one displacement loop, the size of the initiation sequence in such molecules should be twice as long if synthesis of the strand is limited by the free energy of superhelix formation. An identical array of size classes of 7 S strands is obtained from this cell line as compared to mouse LA9 cells. This indicates that the extent of 7 S mitochondrial DNA synthesis is most likely determined by a nucleotide sequence specific event.     

Initiation of deoxyribonucleic acid replication in a temperature-sensitive mutant of B. subtilis: evidence for a transcriptional step

A mutant of Bacillus subtilis unable to initiate a new round of replication at 45 C has been described. Here we show that inhibition of DNA synthesis in this mutant is reversible and that DNA synthesis is resumed at low temperature, even in the presence of chloramphenicol. Initiation of a new replication cycle thus can occur in the absence of protein synthesis. A thermolabile component required for initiation therefore appears to be synthesized at 45 C in an inactive form and can be activated at 30 C in the presence of an inhibitor of protein synthesis. Although resistant to chloramphenicol, the reinitiation of replication occurring after lowering the temperature is sensitive to rifampin and streptolydigin.     

Initiation rate of adenovirus DNA synthesis in infected cell

A method was developed to determine the rate of viral DNA synthesis initiation in adenovirus 2-infected cells. The initiation of DNA synthesis appeared as the rate-limiting step for accumulation of viral DNA. The multiplicity of infection slightly influenced the rate of synthesis of viral DNA, and only during the linear phase of viral DNA production. The initiation of DNA-synthesis was found to occur preferentially on newly synthesized DNA molecules. These kinetics data and the effect of novobiocin suggested that binding of viral DNA with some enzymatic complexes favored the replication of a minor, active class of adenovirus DNA molecules.     

Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex.

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.     

Viral DNA Synthesis in Cells Infected by Temperature-Sensitive Mutants of Simian Virus 40

Temperature-sensitive mutants of simian virus 40 (SV40) have been classified as those that are blocked prior to viral DNA synthesis at the restrictive temperature, "early" mutants, and those harboring a defect later in the replication cycle, "late" mutants. Mutants of the A and D complementation groups are early, those of the B, C, and BC groups are late. Our results confirm earlier reports that A mutants are defective in a function required for the initiation of each round of viral DNA synthesis. D mutants, on the other hand, continue viral DNA replication at the restrictive temperature after preincubation at the permissive temperature. The length of time required for D function to be expressed at the permissive temperature-after which infection proceeds unabated on shifting of the cultures to the restrictive temperature-is 10 to 20 h. The viral DNA synthesized in D mutants under these conditions progresses in normal fashion through replicative intermediate molecules to mature component I and II DNA molecules.     

Reactivation of chick erythrocyte nuclei in heterokaryons with temperature-sensitive Chinese hamster cells.

Chinese hamster cell line K12 is temperature-sensitive for the initiation of DNA synthesis. K12 cells synchronized by serum deprivation were collected in early G1(G0). Heterokaryons were formed by fusing chick erythrocytes with serum-starved K12 cells through the use of UV-irradiated Sendai virus. At the permissive temperature (36.5 degrees C), erythrocyte nuclei in heterokaryons enlarged, the chromatin dispersed, and erythrocyte nuclei synthesized DNA at about the same time as the K12 nuclei. At the restrictive temperature (41 degrees C), erythrocyte nuclei enlarged, but neither erythrocyte nor K12 nuclei initiated DNA synthesis. When erythrocyte nuclei were fused with Wg-1A cells, the wild-type parent for ts K12 cells, both kinds of nuclei synthesized DNA at 36.5 degrees C and 41 degrees C. Activation of erythrocyte nuclei was inefficient in heterokaryons incubated in low-serum medium. The results indicate that serum factors and a cellular function defined by the K12 mutation are required for activation of chick erythrocyte nuclear DNA synthesis.     

Mitochondrial DNA synthesis in cell cycle mutants of saccharomyces cerevisiae

Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with α factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.     

Multiple Initiation of Bacteriophage T4 DNA Replication: Delaying Effect of Bromodeoxyuridine

Effects of bromodeoxyuridine (BUdR) substitutions in phage T4 DNA on the initial stages of DNA replication were investigated. Electron microscope studies of partially replicated, light (thymidine-containing) T4 DNA revealed the presence of multiple loops and forks. These DNA preparations had no BUdR in either parental or newly synthesized DNA, and the observations thus show that multiple initiation of DNA replication is a normal event in T4 development and is not caused by the presence of BUdR. A comparison of early replicative stages of light and heavy (BUdR-containing) DNA in cells mixedly infected with light and heavy T4 phage showed that early DNA synthesis occurs preferentially on the light template. Heavy and light parental DNA became associated with the protein complex of replicative DNA with equal efficiency, and there was no effect of BUdR on the net rate of DNA synthesis after infection. Newly synthesized DNA from heavy templates sedimented more slowly through alkaline sucrose gradients than did newly synthesized DNA from light templates and appeared to represent fewer replicative regions per molecule. These data indicate that BUdR substitutions in the DNA caused a slight delay in initiation but that replication of heavy DNA proceeded normally once initiated.     

Initiation of DNA replication at a nuclear matrix-attached chromatin fraction

It is still unclear what nuclear components support initiation of DNA replication. To address this issue, we developed a cell-free replication system in which the nuclear matrix along with the residual matrix-attached chromatin was used as a substrate for DNA replication. We found out that initiation occurred at late G1 residual chromatin but not at early G1 chromatin and depended on cytosolic and nuclear factors present in S phase cells but not in G1 cells. Initiation of DNA replication occurred at discrete replication foci in a pattern typical for early S phase. To prove that the observed initiation takes place at legitimate DNA replication origins, the in vitro synthesized nascent DNA strands were isolated and analyzed. It was shown that they were enriched in sequences from the core origin region of the early firing, dihydrofolate reductase origin of replication ori-beta and not in distal to the origin sequences. A conclusion is drawn that initiation of DNA replication occurs at discrete sub-chromosomal structures attached to the nuclear matrix.     

The Escherichia coli dnaJ mutation affects biosynthesis of specific proteins, including those of the lac operon.

Temperature-sensitive dnaJ mutants of Escherichia coli showed a thermosensitive defect in the synthesis of beta-galactosidase. Synthesis of the lac mRNA was greatly reduced at the restrictive temperature. The mutants were also conditionally defective in the synthesis of a subset of membrane proteins such as succinate dehydrogenase, whereas the synthesis of anthranilate synthetase, encoded by trpED, as well as that of most cellular proteins, was unaffected at the restrictive temperature. The defect was specific for the dnaJ mutants among several dna mutants which are known to be involved in the initiation of DNA synthesis: dnaK, dnaA, and dnaB mutants synthesized each of these proteins normally even at the restrictive temperature. At the restrictive temperature, growth of the dnaJ mutants was arrested at a specific stage of the cell cycle.     

Postreplication repair of deoxyribonucleic acid and daughter strand exchange in uvr- mutants of Bacillus subtilis

The fate of pyrimidine dimers in deoxyribonucleic acid (DNA) newly synthesized by Bacillus subtilis after ultraviolet irradiation was monitored by use of a damage-specific endonuclease that introduces single-strand breaks adjacent to nearly all of the dimer sites. Two Uvr- strains, one defective in the initiation of dimer excision and the other defective in a function required for efficient dimer excision, were found to be similar to their wild-type parent in the kinetics and extent of converting low-molecular-weight DNA newly synthesized after ultraviolet irradiation to high molecular weight. In the Uvr- strains large molecules of newly synthesized DNA remained susceptible to nicking by the damage-specific endonuclease even after extended incubation in growth medium, whereas the enzyme-sensitive sites were rapidly removed from both preexisting and newly synthesized DNA in Uvr+ cells. Our results support the hypothesis that postreplication repair in bacteria includes recombination between dimer-containing parental DNA strands and newly synthesized strands.     

Enhancement of the synthesis of specific cellular polypeptides in a temperature-sensitive Chinese hamster cell line (K12) defective for entry into S phase

Temperature-sensitive Chinese hamster cells (K12) have been shown to be defective for the initiation of new rounds of DNA replication when incubated at the restrictive temperature (40.5 degrees). By temperature shift experiments with synchronous cultures, we have marked out the step at which the mutation is expressed as the four hours preceding the initiation of DNA synthesis. The block imposed by the mutation has been shown to be irreversible. In order to approach the biochemical characterization of the temperature-sensitive function in K12 cells, we have analyzed the cellular proteins synthesized under permissive (35 degrees) and restrictive temperatures. The synthesis of three polypeptides is markedly enhanced when K12 cells are incubated at 40.5 degrees. One of them (band B) has turned out to be a useful biochemical marker of the expression of K12 mutation since its synthesis is not affected in other ts-mutants or in hybrids in which K12 mutation is complemented. In addition, the alteration in band B synthesis is irreversible and occurs during the same stage of the cell cycle at which the mutated function is expressed.     

Single-stranded bacteriophage T5 stO DNA as template for in vitro fMet-tRNA binding to ribosomes and protein synthesis.

Good evidence is provided that fMet-tRNA binding and aminoacid incorporation, when single-stranded DNA is used instead of mRNA in an E. coli cell-free system, are strictly dependent on the magnesium concentration. Ten sites homologous to the initiation sites of translation can be detected on denatured T5 stO DNA when using ribosomes and initiation factors from uninfected E. coli F. In S-30 extracts, at high magnesium concentrations and in the presence of neomycin, initiation of the translation of denatured T5 stO DNA begins anywhere on the molecule, and yet high molecular weight polypeptides are synthesized. The template potentiality of the denatured T5 stO DNA decreased when using ribosomes plus initiation factors and crude extracts from T5 stO-infected bacteria. By in vitro formation of initiation complexes sites analogous to initiation sites of translation were localized on T5 stO DNA molecules using single-stranded fragments separated by sedimentation in alkaline sucrose gradient.