Complete replication of templates by Escherichia coli DNA polymerase III holoenzyme

DNA polymerase III holoenzyme (holoenzyme) processively and rapidly replicates a primed single-stranded DNA circle to produce a duplex with an interruption in the synthetic strand. The precise nature of this discontinuity in the replicative form (RF II) and the influence of the 5' termini of the DNA and RNA primers were analyzed in this study. Virtually all (90%) of the RF II products primed by DNA were nicked structures sealable by Escherichia coli DNA ligase; in 10% of the products, replication proceeded one nucleotide beyond the 5' DNA terminus displacing (but not removing) the 5' terminal nucleotide. With RNA primers, replication generally went beyond the available single-stranded template. The 5' RNA terminus was displaced by 1-5 nucleotides in 85% of the products; a minority of products was nicked (9%) or had short gaps (6%). Termination of synthesis on a linear DNA template was usually (85%) one base shy of completion. Thus, replication by holoenzyme utilizes all, or nearly all, of the available template and shows no significant 5'----3' exonuclease action as observed in primer removal by the "nick-translation" activity of DNA polymerase I.     

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.     

Adenovirus terminal protein protects single stranded DNA from digestion by a cellular exonuclease.

Adenovirus 5 DNA-protein complex is isolated from virions as a duplex DNA molecule covalently attached by the 5' termini of each strand to virion protein of unknown function. The DNA-protein complex can be digested with E. coli exonuclease III to generate molecules analogous to DNA replication intermediates in that they contain long single stranded regions ending in 5' termini bound to terminal protein. The infectivity of pronase digested Adenovirus 5 DNA is greatly diminished by exonuclease III digestion. However, the infectivity of the DNA-protein complex is not significantly altered when up to at least 2400 nucleotides are removed from the 3' ends of each strand. This indicates that the terminal protein protects 5' terminated single stranded regions from digestion by a cellular exonuclease. DNA-protein complex prepared from a host range mutant with a mutation mapping in the left 4% of the genome was digested with exonuclease III, hybridized to a wild type restriction fragment comprising the left 8% of the genome, and transfected into HeLa cells. Virus with wild type phenotype was recovered at high frequency.     

Degradation of linear DNA by a strand-specific exonuclease activity in Xenopus laevis oocytes.

Linear DNA injected into Xenopus laevis oocyte nuclei recombines with high efficiency if homologous sequences are present at overlapping molecular ends. We found that injected linear DNA was degraded by a 5'----3' strand-specific exonuclease activity during incubation in the oocyte nucleus to leave a heterogeneous population of 3'-tailed molecules. Decreasing the concentration of DNA injected increased the heterogeneity and the average rate of degradation. The 3' tails created were relatively stable; among molecules persisting after overnight incubation, many had 3' tails intact to within 10 bases of the original ends. DNA molecules that were efficient substrates for homologous recombination in oocytes were also partially degraded, leaving 3' tails. We found no evidence for other potent nuclease activities. If molecules with recessed 3'-OH ends were injected, endogenous polymerase efficiently resynthesized complementary strands before degradation of the 5' tails occurred. 3'-tailed molecules are plausible intermediates in the initiation of homologous recombination events in Xenopus oocyte nuclei.     

Action of ATP-dependent DNase from Hemophilus influenzae on cross-linked DNA molecules.

The ATP-dependent DNase from Hemophilus influenzae digests double-stranded linear DNA molecules exonucleolytically while hydrolyzing large amounts of ATP to ADP. Various cross-linked linear duplex DNA molecules are partially resistant to the exonuclease action. Vaccinia DNA, containing natural terminal cross-links (probably in the form of terminal single-stranded loops), is much more slowly degraded than comparable "open-ended" DNA molecules, and ATP is consumed at a proportionately lower rate. It is postulated that the vaccinia DNA molecules undergo slow terminal cleavage by the single strand specific endonuclease activity of the enzyme, and are then rapidly degraded by the double strand exonuclease activity. Phage T7 DNA, containing an average of 100 4',5'8-trimethylpsoralen cross-links/molecule at random internal sites, is digested only to the extent of 2 to 3%. However, ATP hydrolysis continues at a linear rate long after DNA digestion has ceased. A stable enzyme-DNA complex is formed as demonstrated by co-sedimentation of DNA and ATPase activity in sucrose gradients. The hypothesis is advanced that the enzyme digests exonucleolytically to the first cross-link at each end of the DNA molecules where further movement is prevented. The enzyme then remains bound at the cross-links and functions continuously as an ATPase.     

Characteristics of the nascent and non-nascent small DNA molecules found in Escherichia coli

We have purified nascent DNA molecules from Escherichia coli pulse-labeled with 5-bromo[6-3H]deoxyuridine by repeated chromatography on nitrocellulose and isopycnic centrifugation in CsCl. The nascent molecules were labeled with 32P either at their 5' ends using polynucleotide kinase or at their 3' ends using terminal transferase. Compared to the non-nascent DNA of normal density, the nascent dense DNA contained a higher proportion of molecules terminated at their 5' ends with ribonucleotides. Exposure of the dense DNA to alkali generated 5' OH termini quantitatively equivalent to the number of molecules bearing 5' ribonucleotides. Experiments designed (1) to detect structures at the 5' ends of phosphatase-treated nascent DNA molecules that caused them to be resistant to hydrolysis by spleen exonuclease or (2) to detect polypeptides that were associated covalently with small DNA molecules and could be iodinated with the Bolton-Hunter reagent did not yield positive results. We conclude that many, if not all, of the intermediates in E. coli DNA replication are initiated with one or more ribonucleotides. The nascent molecules are outnumbered by small non-nascent DNA molecules in the cell, many of which appear to become slightly longer when cells are pulsed with thymidine. Many of the non-nascent DNA molecules behave as if they were self-complementary or crosslinked.     

Identification and characterization of a protein covalently bound to DNA of minute virus of mice.

We identified a protein which is covalently linked to a fraction of the DNA synthesized in cells infected with minute virus of mice. This protein is specifically bound to the 5' terminus of the extended terminal conformers of the minute virus of mice replicative-form DNA species and of a variable fraction of single-stranded viral DNA. The chemical stability of the protein-DNA linkage is characteristic of a phosphodiester bond between a tyrosine residue in the protein and the 5' end of the DNA. The terminal protein (TP) bound on all DNA forms has a relative molecular weight of 60,000; it is also seen free in extracts from infected cells. Immunologic comparison of the TP with the other known viral proteins suggests that the TP is not related to the capsid proteins or NS-1.     

Sequence analysis of the termini of virion and replicative forms of minute virus of mice DNA suggests a modified rolling hairpin model for autonomous parvovirus DNA replication.

The nucleotide sequences of the terminal regions of monomer replicative form DNA, a pivotal intermediate species in the replication of minute virus of mice, were determined. The left (3') terminus had a unique sequence on both strands and in both 3'-hairpin configurations. In contrast, the right (5') terminus was sequence heterogeneous and extended an additional 18 base pairs beyond that expected from the known sequence of the virion DNA. These data unambiguously establish the sequence complexity at the termini of both the single-stranded viral genome and the pool of replicative DNA. A comparison of the combined sequence information leads us to propose a modified rolling hairpin model for the replication of autonomous parvoviruses which is compatible with all available data.     

Protein n, a primosomal DNA replication protein of Escherichia coli. Purification and characterization

Protein n, essential in forming the primosome for the in vitro conversion of phi X174 single-stranded (SS) DNA to the duplex replicative form (RF), has been purified about 5000-fold to near homogeneity from Escherichia coli. Protein n is heat- and acid-resistant and N-ethyl-maleimide-sensitive. It appears to be a dimer of 12,000 (+/- 2000)-dalton polypeptides. About 80 molecules of protein n are present/cell. Protein n binding to phi X SS DNA depends on the presence of single-strand binding protein (SSB). This requirement for SSB reflects a direct interaction of protein n and SSB. About 30 protein n monomers can be bound to an SSB-coated circle. However, in forming the primosome on an SSB-coated phi X circle, an input of only 2-3 protein n monomers is required and 1 monomer bound/circle. Retention of this low level of protein n on SSB-coated phi X SS DNA is dependent upon protein n', a DNA-dependent ATPase (dATPase) that guides primosome assembly. This single protein n monomer is retained in the assembled primosome, which is conserved on the completed parental RF and participates in the next stage of the replicative cycle, production of progeny RF.     

An electron microscope study of the proteins attached to polio virus RNA and its replicative form (RF).

A recently described method (Wu, M. and Davidson, N. (1978), Nucleic Acids Research 5, in press) for visualizing proteins attached to nucleic acids in the electron microscope has been applied to study proteins attached to poliovirion RNA and to the viral double-stranded intracellular RF form. A protein is found at the 5' end of the plus strand virion RNA, and protein components are found at both ends of the duplex RF. In the RF as usually extracted, there is frequently a larger or compound protein aggregate at the end which contains the 3' end of the plus strand and the 5' end of the minus strand. Banding in CsCl-guanidinium hydrochloride in the presence of sarkosyl causes dissociation of some components of this aggregate, leaving both ends labeled with the covalently bound VPg. These results confirm and extend previous biochemical studies of proteins bound to poliovirion RNA and to the RF form.     

Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae

An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.     

DNA sequence analysis. Terminal sequences of bacteriophage phi80.

Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA have been determined. Sequences of the cohesive ends were obtained through the use of two standard methods. The first method involved the incorporation of all four labeled deoxyribonucleotides into the phi80 cohesive ends using DNA polymerase I. The DNA was then partially digested with micrococcal nuclease or pancreatic DNase. The products were separated by two-dimensional electrophoresis and characterized by composition, 3'-terminal, and nearest neighbor analyses. The second method involved partial incorporation using one, two, or three labeled deoxyribonucleotides followed by similar analyses. Sequences of the double-stranded regions adjacent to the cohesive ends were determined by three new methods. These methods were: (a) the DNA was specifically labeled at the 3' terminus and then partially degraded. Labeled oligonucleotide products were sequenced by their mobilities on various separation systems. (b) The cohesive ends were enlarged by limited degradation with exonuclease III. After this treatment, the DNA was partially repaired with labeled nucleotides, digested, and the products were analyzed. (c) A synthetic ologonucleotide primer was bound to phi80 DNA which had been repaired with DNA polymerase I, and then partially digested with lambda-exonuclease. The primer was extended into the region of interest by partial repair with labeled nucleotides. The extended primer was isolated and analyzed.     

Mechanism of replication of bacteriophage phi X174 XIX. Initiation of phi X174 viral strand DNA synthesis at internal sites on the genome.

Bacteriophage phi X174 viral strand DNA molecules shorter than genome length found late in the infectious cycle in Escherichia coli were 5' end labeled with 32P. Hybridization of the 32P-labeled molecules to restriction enzyme fragments of phi X replicative form DNA revealed an excess of phi X molecules whose 5' ends mapped in HaeIII fragments Z3 and Z4 in comparison with fragments Z1 and Z2. This suggests that initiation of phi X174 viral strand DNA synthesis may occur at internal sites on the complementary strand. There are several appropriately located sequences that might serve as n' (factor Y) recognition sequences and thereby facilitate discontinuous synthesis of the viral strand.     

The genome of lipid-containing bacteriophage PRD1, which infects gram-negative bacteria, contains long, inverted terminal repeats.

The bacteriophage PRD1 is a lipid-bearing phage that infects a wide variety of gram-negative bacteria, including Escherichia coli and Salmonella typhimurium when they contain the appropriate plasmid. It contains a linear duplex DNA molecule that is covalently bound by its 5' ends to a terminal protein. We report here that the PRD1 genome contains a 111-base-pair terminal inverted repeat which does not bear homology to that of any known linear duplex DNAs with terminal proteins. We further report that its 3' termini are susceptible to enzymatic digestion by exonuclease III.     

Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro.

Replication of bacteriophage phi 29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism. In the present paper we have used an in vitro phi 29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. Two types of replicative intermediates were observed: type I (full-length double-stranded phi 29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length phi 29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a single-stranded DNA portion spanning to the other end). Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro phi 29 DNA replication system. Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner. Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded phi 29 DNA molecules were synthesized. In addition, replication of recombinant phi 29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded phi 29 DNA molecules. These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge.     

Hepatitis B virus contains protein attached to the 5′ terminus of its complete DNA strand

Hepatitis B virus DNA contains a tightly bound protein which was not removed by heating to 60°C with 2% SDS, 2% mercaptoethanol. The protein was indirectly demonstrated by the extraction of the DNA-protein complex with phenol before but not after its digestion with proteinase K. The DNA-protein complex had a lower buoyant density than protease-treated or free DNA; it was bound to glass fiber filters; it migrated at a slower rate in gel electrophoresis; and it could be radiolabeled by oxidative iodination. The binding site of the protein was mapped by extraction of restriction endonuclease digests with phenol and analysis of the digests for missing DNA fragments. The protein was localized to a site near the 5′ end of the complete viral DNA strand. It remained attached to this strand after heating with SDS to 90°C or treatment with 0.1 N NaOH, suggesting a covalent linkage. The 5′ end of neither viral DNA strand could be phosphorylated in a reaction with polynucleotide kinase, consistent with attachment of protein to the 5′ ends. The incomplete DNA strand, however, which is the strand elongated by the virion DNA polymerase reaction, did not contain a detectable amount of polypeptide as did the complete strand. The reasons for the apparent block of the 5′ end of the incomplete DNA strand is thus not known. The protein bound covalently to HBV DNA could be involved in the replication of the complete viral DNA strand and/or endonucleolytic generation of linear unit-length DNA pieces from replicative intermediates, although its function and origin are not yet known.     

The structure of poliovirus replicative form.

The structure of polio replicative form (RF) has been investigated by 3' end labeling and the use of polynucleotide phosphorylase to now allow a complete composite of the RF structure. The evidence presented indicates that the 3' terminal sequence of the minus strand is an exact complement to the 5' end of polio RNA. This suggests that the 5' terminal U of polio RNA is genetically coded. Other data is presented to show that in addition to the genetically coded poly(A) tract of the plus strand in RF, a single-stranded poly(A) tail protrudes beyond the double-stranded RNA.