Method of mapping DNA replication origins.

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.     

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA.     

Maturation of replicating simian virus 40 DNA molecules in isolated nuclei by continued bidirectional replication to the normal termination region.

Mature SV40 DNA synthesized for different periods of time either in isolated nuclei or in intact cells was highly purified and then digested with restriction endonucleases in order to relate the time of synthesis of newly replicated viral DNA to its location in the genome. Replication in nuclei supplemented with a cytosol fraction from uninfected cells was a faithful continuation of the bidirectional process observed in intact cells, but did not exhibit significant initiation of new replicons. SV40 DNA replication in cells at 37 degrees C proceeded at about 145 nucleotides/min per replication fork. In the absence of cytosol, when DNA synthesis was limited and joining of Okazaki fragments was retarded, bidirectional SV40 DNA replication continued into the normal region where separation yeilded circular duplex DNA molecules containing one or more interruptions in the nascent DNA strands. In the presence of cytosol, this type of viral DNA was shown to be a precursor of covalently closed, superhelical SV40 DNA, the mature from of viral DNA.     

DNA replication in bacteriophage-infected Staphylococcus aureus.

The synthesis of viral and host DNA in phage-infected Staphylococcus aureus was examined. Three intracellular forms of phage 52HJD DNA were demonstrated: covalently closed circular, open circular, and linear DNA species. It was noted that infection of S. aureus-propagating strains 81 and 52 with phage 52HJD inhibited the replication of the bacterial chromosome and a stringently controlled penicillinase plasmid. A small tetracycline plasmid, normally under relaxed replication control, continued to replicate in the postinfection period. No breakdown of the host chromosome into small-molecular-weight fragments or utilization of bacterial DNA material for the synthesis of viral DNA was observed.     

Replication of polyoma DNA in isolated nuclei: analysis of replication fork movement.

The movement of replication forks during polyoma DNA synthesis in isolated nuclei was analyzed by digesting newly synthesized DNA with the restriction endonuclease HpaII which cleaves polyoma DNA into eight unique fragments. The terminus of in vitro DNA synthesis was identified by cleaving newly completed molecules with HpaII. The distribution of label in the restriction fragments showed that the in vitro DNA synthesis was bidirectional and had the normal terminus of replication. Analysis of replicative intermediates pulse-labeled in vitro further suggested that DNA synthesis in isolated nuclei is an ordered process similar to replication in intact cells. Replication forks moved with a constant rate from the origin towards the terminus of replication. The nonlinear course of the DNA synthesis reaction in the isolated nuclei seems to result from the random inactivation of replication forks rather than a decrease in the rate of fork movement. During the in vitro synthesis a replication fork could maximally synthesize a DNA chain about 1,000 nucleotides long. The results suggest that some replication forks might be initiated in vitro at the origin of replication.     

DNA bending near the replication origin of IncFII plasmid NR1.

The DNA replication origin of plasmid NR1 is located approximately 190 base pairs downstream from the 3' end of the repA1 gene, which encodes the essential initiation protein for replication of the plasmid. Restriction endonuclease fragments that contain the NR1 replication origin and its flanking sequences at circularly permuted positions were obtained by digesting oligomers of ori-containing DNA fragments with sets of enzymes that each cut only once in every ori fragment. Polyacrylamide gel electrophoresis of these permuted restriction fragments showed anomalous mobilities, indicating the presence of a DNA bending locus. Through analysis of the relative mobility plots of these permuted fragments, we found one or two possible DNA bending sites located in the intervening region between the repA1 gene and the replication origin of NR1. It seems possible that DNA bending in this region might help to orient the replication origin alongside the repA1 gene, which could contribute to the cis-acting character of the RepA1 initiation protein.     

In vitro termination of adenovirus DNA synthesis by a soluble replication complex.

The double-stranded DNA from a soluble DNA replication complex that was labeled with deoxyribonucleoside triphosphates and completed in vitro was digested with EcoRI, Sma I, and Hpa I restriction endonucleases. All regions of the adenovirus type 2 genome were labeled in vitro, but restriction fragments derived from the ends of the DNA molecules were relatively more highly labeled than those derived from internal regions. The in vitro endogenous DNA polymerase reaction also exhibited strand-specific labeling near the molecular ends, in that restriciton fragments from the left end were labeled predominantly in the r strand and fragments from the right end were labeled predominantly in the l strand.     

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.     

Evidence for multiple vegetative DNA replication origins and alternative replication mechanisms of bovine papillomavirus type 1

By following up the chance detection in the electron microscope of a DNA replication intermediate within a preparation of bovine papillomavirus (BPV-1) DNA isolated from purified virus particles, information was obtained about the mechanism of BPV-1 genome replication during the final stages of virus multiplication in naturally infected bovine wart tissue. The structure of viral replication intermediates was investigated by electron microscopic analysis of viral DNA linearized by digestion with restriction endonucleases which cleave the circular BPV-1 chromosome at defined sites. Both Cairns and rolling circle-type molecules were identified. Furthermore, replication eyes were widely distributed within the viral genome, indicating that vegetative BPV-1 DNA replication origins are largely uncoupled from previously described plasmid maintenance sequence elements.     

Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis

Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.     

Initiation of adenovirus DNA replication.

In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared with those obtained in a soluble nuclear extract competent for viral DNA replication. It was observed that in vitro DNA replication, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, occurs in a manner apparently indistinguishable from the situation in virus-infected cells. This includes the presence of proteinaceous material on the molecular termini of newly initiated viral DNA.     

Single-stranded replication intermediates of ribosomal DNA replicons of pea.

Replication of ribosomal DNA replicons in cells of Pisum sativum (cv. Alaska) occurs bidirectionally by displacement loops. Replication is initiated on opposite parental strands and nascent chains are elongated moving 5'----3' along each parental template. Replicative intermediates were analyzed by 2-dimensional agarose gel electrophoresis under neutral--neutral and neutral--alkaline conditions. Southern blots of ribosomal DNA fragments separated in the second dimension under neutral conditions show slowly migrating replicative fragments that hybridize with specific probes in a manner consistent with bidirectional replication. The replicative fragments are present in root meristems with cells in S phase; they are absent or few in number in meristems with cells in G2 phase. The following observations indicate that the replicative fragments are single stranded. The apparent length of the replicative fragments is not the same when separated under neutral and alkaline conditions. They contain rDNA without breaks and they do not exhibit the smaller nascent chains expected from replication bubbles and forks. They are not cleaved by restriction enzymes that require duplex DNA as substrate and they are digestible by S1 nuclease.     

Structure of an intermediate in the replication of bacteriophage φX174 deoxyribonucleic acid: The initiation site for DNA replication

Replicative form DNA composed of a closed complementary strand and a discontinuous viral strand has been isolated from cells infected with bacteriophage φX174 during the period of single-strand DNA synthesis. This RFII DNA was degraded by the restriction enzyme from Hemophilus influenzae, endonuclease R, and the products analyzed by polyacrylamide gel electrophoresis. The results indicate that there are two types of discontinuity in the viral strands of these molecules: (1) 65% of the molecules contain a gap, which causes a discrete increase in mobility of a specific restriction enzyme fragment, R3. This gap can be selectively repaired with Escherichia coli DNA polymerase I and nucleoside triphosphates, but the molecules are not converted to RFI by addition of E. coli polynueleotide ligase to the reaction mixture. Approximately 30 moles of radioactive TTP are incorporated per mole of RF DNA. (2) 35% of the RF molecules contain a discontinuity, which does not result in a detectable change in mobility of any restriction enzyme fragment. These RF molecules can be converted to RFI by the action of ligase and polymerase I in the presence of nucleoside triphosphates, with incorporation of only approximately one mole of radioactive TTP, specifically into fragment R3, per mole of RF DNA.When the reaction of late RFII DNA and polymerase I is allowed to proceed beyond the repair of the discontinuity, radioactive nucleotides are incorporated into endonuclease R fragments adjacent to R3 in the 5′ → 3′ direction. This technique was utilized to determine a partial order of endonuclease R fragments in φX174.These results suggest that the synthesis of single-strand DNA is initiated from a unique point in cistron A and proceeds clockwise round the φX174 genetic map (cistron order: ABCDEFGH). A comparison of these results with other studies on φX174 suggests that DNA synthesis in all stages of φX174 replication may be initiated from a specific locus on the genome, at or near cistron A.     

Bidirectional replication of adenovirus type 2 DNA.

After short periods of labeling with [3H]thymidine, recently completed adenovirus DNA molecules were isolated and cleaved with restriction endonucleases. The strands (heavy and light) of most of the restriction endonuclease fragments were separated. The pattern of labeling clearly shows an asymmetry of radioactivity on the isolated strands of each restriction endonuclease piece. The data is consistent with replication proceeding in the 5' to 3' direction on each strand. Thus, there is an initiation point placed at or near each end of the molecule.     

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.     

Tracking bacterial DNA replication forks in vivo by pulsed field gel electrophoresis.

The location of chromosomal DNA replication forks was identified in synchronously replicating E. coli cultures by pulse labeling DNA at specific times with 14C-thymidine and following incorporation of radionucleotide into genomic Not I restriction fragments. This technique could be used to characterize chromosomal DNA replication, to characterize mutations which affect this process, to identify the location of DNA replication origins and termini as well as aid in the construction of macrorestriction maps. Here, we further characterize the DNA replication mutations divE and dnaK and preliminary characterize the genomic organization of E. coli isolate 15.     

Presence of protein at the termini of intracellular adenovirus type 5 DNA.

Adenovirus type 5 contains linear double-stranded DNA with protein covalently attached to the ends of the molecules. The presence of protein at the termini of intracellular viral DNA in adenovirus type 5-infected cells was investigated at different stages during the replication process. The intracellular viral DNA was isolated from the nuclei by lysis in 4 M guanidine hydrochloride. Electrophoresis on agarose gels of HsuI restriction enzyme fragments and sucrose gradient centrifugation were used to detect protein on intracellular viral DNA. After uncoating parental DNA still contains protein attached to the termini of the viral genome. Replicating and mature progeny viral DNA can also be isolated in the form of DNA-protein complexes. These complexes exhibit the same properties as the DNA-protein complex isolated from purified virions. These results suggest that the protein at the termini of intracellular viral DNA is identical to the protein attached to the 5'-ends of the DNA extracted from virions and that it is possibly involved in the replication of viral DNA.     

The replication origin of proplastid DNA in cultured cells of tobacco

Summary When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.     

A preparation of short DNA chains synthesized in vivo after introduction into DNA from live cells of trioxsalen crosslinks is not enriched by replication initiation regions

The experiments undertaken in order to verify the trioxsalen-crosslinking method suggested by Russev and Vassilev for isolation of eukaryotic replication origins are described. It was found that the preparation of viral DNA isolated by the above mentioned method from CV-1 cells lytically infected with SV40 was not enriched in sequences including SV40 replication origin. The hybridization pattern of DNA preparation isolated by the trioxsalen-crosslinking procedure from chicken erythroblastosis cells with the cloned fragments of globin gene domain was found to be identical to those of the total DNA probe. The DNA fraction enriched in replication origins was isolated from the same cells with the aid of nascent DNA strand extrusion method by Zannis-Hadjopoulos et al. The hybridization pattern of this DNA fraction with the cloned fragments of chicken alpha-globin gene domain was different from those of total DNA. Taking together, the results of our experiments demonstrate that trioxsalen-crosslinking procedure does not lead to the isolation of replication origins from the objects studied in the present investigation.