Precise localization of the origin of replication in a physical map of HeLa cell mitochondrial DNA and isolation of a small fragment that contains it

The precise positions of the origin of replication³ and of the D-loop within the HpaII restriction map of HeLa cell mitochondrial DNA have been investigated. For this purpose, 7 S DNA, which is the heavy-chain initiation sequence, was used as a template for fragment-primed DNA synthesis by Escherichia coli DNA polymerase I. The results indicate clearly that the origin of replication lies in HpaII fragment 8 at about 80 base-pairs from the border with fragment 17, and that the D-loop region extends from this site, through fragment 17, to a position in fragment 10 which is about 365 base-pairs from the border with fragment 17. Sequential digestion of fragment 8 with HaeIII enzyme has allowed the isolation of a subfragment, about 200 base-pairs long, that contains the origin of replication.     

Studies on the mechanism of enzymatic DNA elongation by Escherichia coli DNA polymerase II.

The mechanism of enzymatic elongation by Escherichia coli DNA polymerase II of a DNA primer, which is annealed to a unique position on the bacteriophage fd viral DNA, has been studied. The enzyme is found to dissociate from the substrate at specific positions on the genome which act as “barriers” to further primer extension. It is believed these are sites of secondary structure in the DNA. When the template is complexed with E. coli DNA binding protein many of these barriers are eliminated and the enzyme remains associated with the same primer-template molecule during extensive intervals of DNA synthesis. Despite the presence of E. coli DNA binding protein, at least one barrier on the fd genome remains rate-limiting to chain extension and disturbs the otherwise processive mechanism of DNA synthesis. This barrier is overcome by increasing the concentration of enzyme.In contrast, it is found that DNA polymerase I is not rate-limited by structural barriers in the template, however, it exhibits a non-processive mechanism of elongation.These findings provide a framework for understanding the necessity for participation of proteins other than a DNA polymerase in chain extension during chromosomal replication.     

A rolling circle model of the in vivo replication of bacteriophage phiX174 replicative form DNA: different fate of two types of progeny replicative form

In a preceding paper (Schröder and Kaerner, 1972) a rolling circle mechanism has been described for the replication of bacteriophage φX174 replicative form. Replication involved nicking and elongation of the viral (positive) strand component of the RF molecule resulting in the displacement of a single-strand tail of increasing length. The synthesis of the new complementary (negative) strand on the single-strand tails appears to be initiated with considerable delay and converts the tail into double-stranded DNA. Before the new negative strand is completed the replicative intermediates split into (I) a complete RF molecule containing the “old” negative and the new positive strand, and (II) a linear, partially double-stranded “tail” consisting of the complete old positive strand and a fragment of the new negative strand.The present study is concerned with the fate during RF replication of these fragments of the rolling circles. Those RFII molecules containing the old negative strands appear to go into further replication rounds repeatedly. Some of the tails were found in the infected cells in their original linear form. “Gapped” RFII molecules, which have been described earlier by Schekman and co-workers (Schekman &; Ray, 1971; Schekman et al., 1971), are supposed to originate from the tails of rolling circle intermediates by circularization of their positive strand components. Evidence is provided by our experiments that even late during RF replication these gaps are present only in the negative strands of RFII. Appropriate chase experiments indicated that the tails finally are converted to RFI molecules. Progeny RFI molecules could not be observed to start new replication rounds under our conditions although we cannot exclude that this might happen to some minor extent.The results presented suggest that the master templates for RF replication are the first negative strands to be formed, rather than the parental positive strands.     

Lack of repair of ultraviolet light damage in Mycoplasma gallisepticum

Molecules with single-stranded tails (rolling circles) were isolated as replicating intermediates in G4 progeny single-stranded DNA synthesis. Lysates from infected cells harvested late in infection during single-stranded DNA synthesis were not deproteinised but analysed directly in caesium chloride and propidium diiodide gradients. The gradient fractionated them on the basis of tail length. If the lysates were first deproteinised however, the tailed replicative intermediates banded as a peak at a density just greater than that of replicative form II DNA (RFII) and did not spread down the gradient. The origin of synthesis of the viral strand tail was mapped by electron microscopy as 55 to 60% away from the single EcoRI cleavage site. Termination molecules finishing a round of viral strand DNA synthesis have been identified as molecules consisting of a closed single-stranded DNA circle attached by a very small region to the parent double-stranded DNA circle.     

Mechanism of viral replication. I. Structure of replication complexes of R17 bacteriophage

The replication of R17 bacteriophage in Escherichia coli MRE-600 cells was investigated using a new electron microscopic technique. The structures of replicating ribonucleoprotein complexes, as well as of purified replicative intermediate and replicative form, were studied. These structures are identical to those predicted by the model of Weissmann et al. (1968). From this it may be concluded that replication proceeds through essentially single-stranded inter-mediates and that double-stranded structures are either by-products or artifacts.     

G4 DNA replication: I. Origin of synthesis of the viral and complementary DNA strands

The break in the complementary DNA strand of early G4 replicative form II DNA (RFII) and in the viral DNA strand of late RFII DNA was located using two single cleavage restriction enzymes (EcoRI and PstI) and by limited nick translation of the break using DNA polymerase I and ³²P-labelled deoxyribonucleotides followed by digestion with the restriction enzymes HaeIII and HindII. The break in the complementary DNA strand was unique and in HaeIII Z5 close to the EcoRI cleavage site whereas the break in the viral DNA strand was on the other side of the molecule in HaeIII Z2 approxiately 50% away from the EcoRI cleavage site. Distribution of a short ³H pulse in early G4 replicating intermediates that were synthesising both DNA strands at the same time showed that synthesis of the strands started on opposite sides of the molecule and proceeded in opposite convergent directions, suggesting that initiation of synthesis of the two strands was independent and not unified in a single growing fork.     

Comparison of partial denaturation maps with the known sequence of simian virus 40 and phi X174 replicative form DNA.

Electron microscope partial denaturation maps of two viral DNAs, simian virus 40 and φX174 replicative form, have been obtained. A simple computer program has been developed to predict denaturation maps from any given DNA sequence, based on the percentage of A · T base-pairs along the molecule. Maps constructed from the SV40 DNA and φX174 replicative form DNA base sequence show a good correlation with the experimental maps. The results show that the regions of a DNA molecule that denature first are, in fact, those regions with the highest content of adenine and thymine base-pairs.     

Transcriptional organization of the Drosophila melanogaster ribosomal RNA genes.

Five distinct DNA replicating intermediates have been separated from lysates of bacteriophage G4-infected cells pulse-labelled during the period of replicative form synthesis using propidium diiodide/caesium chloride gradients. These are a partially single-stranded theta structure that is labelled in both the viral and complementary DNA strands; partially single-stranded circles, some with an unfinished viral DNA strand (25%) and some with an unfinished complementary DNA strand (75%); replicative form II(RFII) and replicative form I(RFI) DNA labelled only in the complementary DNA strand. To explain the pulse-label data a model is proposed in which G4 replicative form replication takes place by a displacement mechanism in which synthesis of the new viral DNA strand displaces the old viral DNA strand as a single-stranded DNA loop (D-loop) and when the displacement reaches half way round the molecule (the origin of synthesis of the G4 viral and complementary DNA strands are on opposite sides of the genome, Martin &; Godson 1977) synthesis of the complementary DNA strand starts, but in the opposite direction. Strand separation of the parent helix runs ahead of DNA synthesis, releasing two partially single-stranded circles from the replicating structure which then complete their replication as free single-stranded DNA circles. No evidence was found to support a rolling circle displacement mechanism of G4 replicative form synthesis.     

Construction of an M13 histidine-transducing phage: a single-stranded cloning vehicle with one EcoRI site.

In order to create a ready source of single-stranded DNA for DNA sequence determination by the dideoxy chain-termination method, the promoter-proximal part of the histidine operon, the hisOGD region of Salmonella typhimurium, was cloned onto the single-stranded phage M13. Both orientations of the his DNA were cloned to supply DNA template for sequencing of each strand. Insertion was achieved at an HaeIII site in the intergenic region (IR) of M13, and a single EcoRI site was purposely regenerated at one boundary of the his DNA insert. Infected colonies, not plaques, were selected using the hisD gene as a selective marker. The single RI site and the hisD marker for auxotrophic selection represent improvements on the wild type M13 as a single-stranded vector for cloning other DNA.     

Studies on biosynthesis of tobacco mosaic virus. VII. Radioactivity of plus and minus strands in different forms of viral RNA after labelling of infected tobacco leaves

Tobacco leaves were labelled with tritiated undine for 30 or 120 minutes at different times after systemic infection with tobacco mosaic virus. RNA was extracted and separated into three fractions: one enriched in RF (replicative form), one enriched in RI (replicative intermediate), and one containing the bulk of single-stranded RNA. Radioactivity in plus strands (viral RNA) and minus strands (complementary RNA) was determined in each fraction by an isotope dilution assay. The amount of minus strands in the RP and RI fractions and the amount of plus strands in the single-stranded RNA fraction were also determined.Minus-strand synthesis was twice as high a few hours after the outbreak of visible symptoms as during the subsequent large accumulation of plus strands. At the early stage of virus production, the specific radioactivity of the minus strands was three- to fourfold that of the total RNA. Later it was about the same as that of the total RNA. As minus strands constitute a constant part of the total RNA at the later stages, this observation suggests that breakdown of minus strands is small.The specific radioactivity of minus strands was the same in corresponding RF and RI fractions. As the turn-over of minus strands appears to be small, a rapid interconversion of the two RNA types is indicated.In RF and RI the radioactivity in plus strands was between 6 and 50 times greater than that in minus strands. The specific radioactivity of plus strands was greater in RF and RI than in the single-stranded RNA, supporting the concept that both RF and RI have a precursor role for viral RNA.     

An analysis of tobacco mosaic virus replicative structures synthesized in vitro

Summary The RNA structures synthesized in vitro by a crude enzyme complex from tobacco mosaic virus (TMV)-infected leaves have been analyzed; the major viral-specific products were similar to TMV-replicative form (RF) and-replicative intermediate (RI) in electrophoretic behavior and ribonuclease sensitivity. Synthesis of these RF-like and RI-like structures neither required nor responded to added viral RNA, but did require all four ribonucleotide triphosphates. Enriched radiolabeled RF-like and RI-like RNA fractions were isolated from non-denaturing agarose gels by electroelution and hybridized to a collection of TMV sequences cloned into bacteriophage M13. Enriched RF-RNA hybridized to sequences of both plus and minus polarity, while enriched RI-RNA hybridized only to inserts of minus polarity, indicating only plus strand synthesis in this fraction. Most of the label incorporated into the plus strand of the enriched RF-RNA was found near the 3-end of this strand, while most of the label incorporated into enriched RI-RNA was found several hundred bases from the 5-end of the plus strand.Paper presented at the first International Congress of Plant Molecular Biology (Savannah, GA, 1985).     

Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

The role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an $\text{E. coli}$ mutant deficient in the 5′ → 3′ exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.     

Replication of bacteriophage f1: A complex containing gene II protein in gene V mutant-infected bacteria

A dense complex has been isolated from bacteria infected with gene V amber mutant f 1 bacteriophage. The major protein in this complex is the f 1 bacteriophage-specific gene II protein. Other proteins in the complex include the f 1 bacteriophage coat protein and proteins which migrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with the f1 bacteriophage-specific gene III, gene IV and X protein. A protein of approximately 20,000 M_r is also present in the complex. Examination of bacteria infected with gene V mutant f1 bacteriophage revealed the complex as a densely staining amorphous body which appears to be associated with the cytoplasmic membrane. Bacteria infected with f1 bacteriophage that contain amber mutations in genes other than gene V do not contain this complex.     

Regulation of coliphage f1 single-stranded DNA synthesis by a DNA-binding protein

Control of single-strand DNA synthesis in coliphage f1 was studied with the use of mutants which are temperature sensitive in gene 2, a gene essential for phage DNA replication. Cells were infected at a restrictive temperature with such a mutant, and the DNA synthesized after a shift to permissive temperature was examined. When cells were held at 42 °C for ten or more minutes after infection, only single-stranded DNA was synthesized immediately after the shift to permissive temperature. This indicated that the accumulation of a pool of double-stranded, replicative form DNA molecules is not an absolute requirement for the synthesis of single-stranded DNA, although replicative form DNA accumulation precedes single-strand synthesis in cells infected with wild-type phage. Cells infected at restrictive temperature with the mutant phage do not replicate the infecting DNA, but do accumulate a substantial amount of gene 5 protein, a DNA-binding protein essential for single-strand synthesis. It is proposed that this accumulated gene 5 protein, by binding to the limited number of replicating DNA molecules formed following the shift to the permissive temperature, acts to prevent the synthesis of double-stranded replicative form DNA, thus causing the predominant appearance of single strands. This explanation implies an intermediate common to both single and double-stranded DNA synthesis. The kinetics of gene 5 protein synthesis indicates that it is the ratio of the gene 5 protein to replicating DNA molecules which determines whether an intermediate will synthesize double or single-stranded DNA.     

Insertion of the Tn3 transposon into the genome of the single-stranded DNA phage M13.

The transposable genetic element Tn3, which carries an ampicillin (Ap) resistance determinant, has been translocated from a ColE1-Apr plasmid, RSF2124, to the genome of the filamentous single-stranded DNA phage M13. The site orientation of the inserted element has been determined for one such phage, M13::Tn3-15. The insertion is within the intergenic space separating genes 2 and 4 and containing both the viral strand and complementary strand origins. The lengths of both the filamentous phage and the duplex replicative form (RF) DNA are 1.7--1.8 times those of M13 phage and replicative form DNA. Both plaque formation and transduction of sensitive cells to ampicillin resistance by M13::Tn3-15 are sensitive to purified antibodies to the M13 major coat protein.