A gap at a unique location in newly replicated kinetoplast DNA minicircles from Trypanosoma equiperdum

Kinetoplast DNA is a network containing thousands of interlocked minicircles. The minicircles replicate as free molecules after release from the network, and their progeny are then reattached (Englund, P. T., (1979) J. Biol. Chem. 254, 4895-4900). In Trypanosoma equiperdum, some of the newly replicated minicircles which have recatenated to the network contain a single gap. This gap is about 10 nucleotides in size and it is in the newly synthesized strand. Based on several criteria (S1 nuclease digestion, denaturing polyacrylamide gel analysis and DNA sequencing), the gap residues at a unique site on the molecule. This site overlaps the sequence GGGGTTGGTGTAA, which is the only common sequence found in all minicircles, from several different species, which have been examined.     

Both strands of polyoma DNA are replicated discontinuously with ribonucleotide primers in vivo

Nascent polyoma DNA molecules were isolated after pulse-labeling of infected murine 3T6 cells with [3H]thymidine. The extent of digestion of these DNA molecules by spleen exonuclease was increased by exposure to alkali or RNase, suggesting that ribonucleotides were present at or near the 5' terminal of the newly synthesized pieces of DNA. Intermediates shorter than 300 nucleotides were hybridized to the separated strands of restriction enzyme fragments of the polyoma genome: 2.5 to 3-fold more radioactivity was found in the strand whose synthesis is necessarily discontinuous (the lagging strand) than in the strand whose synthesis is potentially continuous (the leading strand) than in the strand whose synthesis is potentially continuous (the leading strand). Separation of the strands of [5'-32P]DNA molecules showed that the excess [3H]thymidine in lagging-strand molecules was not simply the result of an increased number of molecules. Therefore, assuming equivalent efficiencies of labeling, lagging-strand pieces must be slightly longer than those with leading-strand polarity. The presence of ribonucleotides on the 5' termini of molecules with both leading- and lagging-strand polarity was demonstrated by (i) release of 32P-ribonucleoside diphosphates upon alkaline hydrolysis of [5'-32P]DNA separated according to replication polarity and (ii) the change in the degree of self-annealing of nascent molecules upon preferential degradation of DNA molecules possessing initiator RNA moieties by spleen exonuclease. We conclude that replication of polyoma DNA in vivo occurs discontinuously on both sides of the growing fork, using RNA as the major priming mechanism.     

Covalent attachment of RNA to nascent DNA in mammalian cells

We have used the technique of phosphate transfer analysis to test for the presence of phosphodiester bonds linking ribonucleotides (on the 5′ side) to deoxyribonucleotides (on the 3′ side) in DNA newly synthesized within lysates or purified nuclei of mammalian cells. We have found that such covalent junctions between RNA and DNA are present at a frequency of one junction per newly synthesized DNA strand. The junctions are located close to the ends of the nascent DNA strands. The stretches of RNA at the junction are very short compared to the stretches of DNA. These properties are consistent with the conclusion by Reichard, Eliasson, and Söderman (1974) that short stretches of RNA are present on the 5′ ends of nascent DNA strands produced during replication of polyoma virus.     

Simian virus 40 DNA replication: characterization of gaps in the termination region.

A class of precursor DNA (pDNA) II molecules has been identified as the immediate precursor of simian virus 40 DNA I. A pDNA II molecule contains a strand of newly synthesized DNA with an interruption located in the region where DNA synthesis terminates (4). These pDNA II molecules have been isolated and further characterized. They are converted to covalently closed structures (simian virus 40 DNA I) only when they are treated in vitro with both T4 DNA polymerase and Escherichia coli ligase. After in vitro repair of pDNA II with T4 DNA polymerase and nucleoside triphosphates, approximately 7 mol of alpha-[32P]dATP is incorporated per mol of DNA II. Alkaline sucrose analysis of these gap-filled molecules, after they have been cleaved with Eco RI restriction endonuclease, has demonstrated that gaps are specifically located in the termination region. alpha-[32P]dATP is incorporated equally into the two labeled products that are generated by RI cleavage of these molecules. This indicates the presence of gaps in both the newly synthesized plus the minus strands. Electrophoretic analysis of the gap-filled molecules, after they have been cleaved with endonuclease Hind, has shown that gaps are localized in Hind fragments G and B and to a minor degree in fragment J. pDNA II molecules have the following properties. There is a gap in the newly synthesized linear DNA strand contained in the pDNA II molecule. Nicked pDNA II molecules cannot be detected. The two molecules that arise by segregation contain gaps in both of the complementary strands. Based on the amount of alpha-[32P]dATP incorporated and the rate of exonuclease III digestion of gap-filled molecules, it is estimated that the size of the gaps is between 22 and 73 nucleotides. Models for termination of DNA synthesis are proposed based on these findings.     

Synthesis and processing of kinetoplast DNA minicircles in Trypanosoma equiperdum.

Kinetoplast DNA, the mitochondrial DNA in trypanosomes, is a giant network containing topologically interlocked minicircles. Replication occurs on free minicircles that have been detached from the network. In this paper, we report studies on the synthesis and processing of the minicircle L and H strands. Analysis of free minicircles from Trypanosoma equiperdum by two-dimensional agarose gel electrophoresis indicated that elongating L strands are present on theta structures. Hybridization studies indicated that L-strand elongation is continuous and unidirectional, starting near nucleotide 805 and proceeding around the entire minicircle. The theta structures segregate into monomeric progeny minicircles, and those with a newly synthesized L strand have a 8-nucleotide gap between nucleotides 805 and 814 (J. M. Ntambi, T. A. Shapiro, K. A. Ryan, and P. T. Englund, J. Biol. Chem. 261:11890-11895, 1986). These molecules are reattached to the network, where repair of the gap takes place. Of the molecules labeled during a 10-min pulse with [3H]thymidine, gap filling occurred on half within about 15 min and on virtually all by 60 min; however, there was no detectable covalent closure of the newly synthesized L strand by 60 min.     

Lagging strand DNA synthesis by calf thymus DNA polymerases alpha, beta, delta and epsilon in the presence of auxiliary proteins.

By using a defined gapped DNA substrate that mimics a lagging strand of 230 nucleotides and that contains a defined pause site, we have analyzed calf thymus DNA polymerases (pol) alpha, beta, delta, and epsilon in the presence of the three auxiliary proteins proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) for their ability to complete an Okazaki fragment. Pol alpha alone could fill the gap to near completion, but was strongly stopped by the pause site. Addition of low amounts of RP-A resulted in an increased synthesis by pol alpha past the pause site. In contrast, high amounts of RP-A strongly inhibited gap filling by pol alpha. Further inhibition was evident when the two other auxiliary proteins, PCNA and RF-C, were added in addition to RP-A. Pol beta could completely fill the gap without specific pausing and also was strongly inhibited by RP-A. PCNA and RF-C had no detectable effect on pol beta. Pol delta, relied as expected, on all three auxiliary proteins for complete gap filling synthesis and could, upon longer incubation, perform a limited amount of strand displacement synthesis. Pol epsilon core enzyme was able to fill the gap completely, but like pol alpha, essentially stopped at the pause site. This pausing could only be overcome upon addition of PCNA, RF-C and E. coli single-stranded DNA binding protein. Thus pol epsilon holoenzyme preferentially synthesized to the end of the gap without pausing. Ligation of the DNA products indicated that pol beta core enzyme, pol delta and pol epsilon holoenzymes (but not pol alpha and pol epsilon core enzyme) synthesized products that were easily ligatable. Our results indicate that pol epsilon holoenzyme fills a defined lagging strand gapped template to exact completion and is able to pass a pause site. The data favour the hypothesis of Burgers (Burgers, P.M.J. (1991) J. Biol. Chem. 266, 22698-22706) that pol epsilon might be a candidate for the second replication enzyme at the lagging strand of the replication fork.     

RNase-dependent discontinuities associated with the crossovers of spontaneously formed joint DNA molecules in Physarum polycephalum

Transient four stranded joint DNA molecules bridging sister chromatids constitute an intriguing feature of replicating genomes. Here, we studied their structure and frequency of formation in Physarum polycephalum. By “3D gels”, we evidenced that they are not made of four continuous DNA strands. Discontinuities, which do not interfere with the unique propensity of the joint DNA molecules to branch migrate in vitro, are linked to the crossover, enhanced by RNaseA, and affect at most half of the DNA strands. We propose a structural model of joint DNA molecules containing ribonucleotides inserted within one strand, a gapped strand, and two continuous DNA strands. We further show that spontaneous joint DNA molecules are short-lived and are as abundant as replication forks. Our results emphasize the highly frequent formation of joint DNA molecules involving newly replicated DNA in an untreated cell and uncover a transitory mechanism connecting the sister chromatids during S phase.     

Size and base composition of RNA segments in complementary strands of supercoiled plasmid ColE1 DNA

Preparations of ColEl plasmid DNA synthesized in the presence of chloramphenicol were separated into samples having gaps resulting from removal of ribonucleotides in one or the other of the complementary DNA strands. These samples were used as templates for repair resynthesis reactions using DNA polymerase of Rous sarcoma virus and α-³²P-labeled deoxyribonucleoside 5′-triphosphates. Reactions involved the incorporation of each labeled nucleotide in the presence of three unlabeled nucleotides, and also the incorporation of all four labeled nucleotides followed by complete digestion and electrophoretic separation of the products. By these two methods the RNA integrated in the light strand of ColEl DNA was found to comprise an average of 38 ribonucleotides with a base composition of 17G, 5A, 8C, and 8U. The RNA segment in the heavy strand consists of an average of 15 ribonucleotides with a base composition of 5G, 2A, 4C, and 4U.     

Position-specific effect of ribonucleotides on the cleavage activity of human topoisomerase II

Beyond the normal DNA transactions mediated by topoisomerase II, we have recently demonstrated that the cleavage activity of the two human topoisomerase II isoforms is several-fold stimulated if a ribonucleotide rather than a deoxyribonucleotide is present at the scissile phosphodiester in one strand of the substrate. Here we show that ribonucleotides exert a position-specific effect on topoisomerase II-mediated cleavage without altering the sequence specificity of the enzyme. Ribonucleotides located within the 4 bp cleavage stagger stimulate topoisomerase II-mediated cleavage, whereas ribonucleotides located outside the stagger in general have an inhibitory effect. Results obtained from competition experiments indicate that the position-specific effect of ribonucleotides on topoisomerase II activity is caused by altered substrate interaction. When cleavage is performed with substrates containing one ribonucleotide in both strands or several ribonucleotides in one strand the effect of the individual ribonucleotides on cleavage is not additive. Finally, although topoisomerase II recognizes substrates with longer stretches of ribonucleotides, an RNA/DNA hybrid where one strand is composed entirely of RNA is not cleaved by the enzyme. The positional effect of ribonucleotides on topoisomerase II-mediated cleavage shares many similarities to the positional effect exerted by either abasic sites or base mismatches, demonstrating a general influence of DNA imperfections on topoisomerase II activity.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Nucleotide assignment of alkali-sensitive sites in mouse mitochondrial DNA

Mature, closed circular mouse mitochondrial DNA contains a significant number of ribonucleotides throughout the genome. Previous studies have implicated the two origins of DNA replication as preferred sites of ribonucleotide retention. We have analyzed the site specificity of ribosubstitution by direct sizing of alkali-treated restriction fragments in comparison with the DNA sequence of untreated restriction fragments of cloned mouse mitochondrial DNA. These results have confirmed the observations that ribonucleotides are retained at the two origins of replication and are most likely remnants of RNA priming events associated with DNA replication. The map location of ribonucleotides at the light strand origin of replication has been refined to a triplet nucleotide (5'-CGG-3') in the light strand initiation region. This approach has demonstrated that all four deoxyribonucleotides are subject to ribosubstitution and no single base (or subset of the four bases) predominates. An examination of selected regions of the mitochondrial DNA genome including the putative coding region for cytochrome oxidase subunit III and regions containing the genes for tRNAPhe, tRNAVal, 12 S rRNA, and 16 S rRNA reveals preferred sites for ribosubstitution. These preferred sites do not relate in any obvious way to the functional aspects of these domains. In addition, the data indicate that every position in the DNA sequences examined can be ribosubstituted at a very low frequency.     

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.     

Isolation and structural characterization of monomeric and dimeric forms of replicative intermediates of Kilham rat virus DNA.

Two virus-specific species of newly synthesized DNA were isolated from rat fibroblast cell cultures infected with the Kilham rat virus (RV). These two DNA species were purified; their behavior on hydroxyapatite chromatography and their sedimentation coefficients in sucrose gradients were determined. One of the two species corresponds to the linear double-stranded form of the RV DNA, and the other corresponds to the dimeric duplex form. After denaturation, a fraction of both species showed an intramolecular renaturation; these molecules are composed of viral strand covalently linked to complementary strand. Models for the structure of both species are posposed. Both species may be considered as double-strand replicative intermediates of the single-stranded RV DNA.     

Nucleotide sequence of cauliflower mosaic virus DNA

The complete nucleotide sequence (8024 nucleotides) of the circular double-stranded DNA of cauli-flower mosaic virus has been established. The DNA molecule is known to possess three discrete single-stranded discontinuities, often referred to as “gaps”, two in one strand and one in the other. The sequence data indicate that gap 1, the single discontinuity in the α strand, corresponds to the absence of no more than one or two nucleotides with respect to the complementary β strand. The two discontinuities in the β strand, however, are not authentic gaps since no nucleotides are missing, but are instead regions of sequence overlap: a short sequence (19 residues for gap 2, at least 2 residues for gap 3) at one terminus of each discontinuity, probably the 5′ terminus, is displaced from the double helix by an identical sequence at the other boundary of the discontinuity. Analysis of the distribution of nonsense codons in the DNA sequence is consistent with other evidence that only the α strand is transcribed. The coding region extends around the circular molecule from 4 map units of gap 1, the map origin, to map position 91, and consists of six long open reading frames. Our findings suggest, but do not prove, that the DNA sequence of the open reading frames is colinear with viral protein sequences. The cistron for the viral coat protein, which is probably synthesized in the form of a precursor, has been situated in coding region IV on the basis of its unusual amino acid composition.     

Discontinuous DNA synthesis by purified mammalian proteins

Five proteins purified from mouse cells acting together efficiently convert a single-stranded circular DNA template to covalently closed duplex circle by a discontinuous mechanism. DNA polymerase alpha/primase with the assistance of alpha accessory factor covers the single-stranded circle with RNA-primed DNA fragments. Primers are removed by a combination of RNase H-1 and a 5'-exonuclease that was identified by its ability to complete this in vitro system. The 5'-exonuclease is required to remove residual one or two ribonucleotides at the primer/DNA junction that are resistant to RNase H-1. Gap filling is by the DNA polymerase alpha/primase, and DNA ligase I converts the DNA fragments to continuous strand. The concerted action of the five proteins emulates synthesis of the staging strand at the replication fork.     

Polymerase mu is a DNA-directed DNA/RNA polymerase

DNA polymerases are defined as such because they use deoxynucleotides instead of ribonucleotides with high specificity. We show here that polymerase mu (pol mu), implicated in the nonhomologous end-joining pathway for repair of DNA double-strand breaks, incorporates both ribonucleotides and deoxynucleotides in a template-directed manner. pol mu has an approximately 1,000-fold-reduced ability to discriminate against ribonucleotides compared to that of the related pol beta, although pol mu's substrate specificity is similar to that of pol beta in most other respects. Moreover, pol mu more frequently incorporates ribonucleotides when presented with nucleotide concentrations that approximate cellular pools. We therefore addressed the impact of ribonucleotide incorporation on the activities of factors required for double-strand break repair by nonhomologous end joining. We determined that the ligase required for this pathway readily joined strand breaks with terminal ribonucleotides. Most significantly, pol mu frequently introduced ribonucleotides into the repair junctions of an in vitro nonhomologous end-joining reaction, an activity that would be expected to have important consequences in the context of cellular double-strand break repair.     

Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.

Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA.     

Gap Filling Activities of Pseudomonas DNA Ligase D (LigD) Polymerase and Functional Interactions of LigD with the DNA End-binding Ku Protein

Many bacterial pathogens, including Pseudomonas aeruginosa, have a nonhomologous end joining (NHEJ) system of DNA double strand break (DSB) repair driven by Ku and DNA ligase D (LigD). LigD is a multifunctional enzyme composed of a ligase domain fused to an autonomous polymerase module (POL) that adds ribonucleotides or deoxyribonucleotides to DSB ends and primer-templates. LigD POL and the eukaryal NHEJ polymerase λ are thought to bridge broken DNA ends via contacts with a duplex DNA segment downstream of the primer terminus, a scenario analogous to gap repair. Here, we characterized the gap repair activity of Pseudomonas LigD POL, which is more efficient than simple templated primer extension and relies on a 5′-phosphate group on the distal gap strand end to confer apparent processivity in filling gaps of 3 or 4 nucleotides. Mutations of the His-553, Arg-556, and Lys-566 side chains implicated in DNA 5′-phosphate binding eliminate the preferential filling of 5′-phosphate gaps. Mutating Phe-603, which is imputed to stack on the nucleobase of the template strand that includes the 1st bp of the downstream gap duplex segment, selectively affects incorporation of the final gap-closing nucleotide. We find that Pseudomonas Ku stimulates POL-catalyzed ribonucleotide addition to a plasmid DSB end and promotes plasmid end joining by full-length Pseudomonas LigD. A series of incremental truncations from the C terminus of the 293-amino acid Ku polypeptide identifies Ku-(1–229) as sufficient for homodimerization and LigD stimulation. The slightly longer Ku-(1–253) homodimer forms stable complexes at both ends of linear plasmid DNA that protect the DSBs from digestion by 5′- and 3′-exonucleases.     

Differential DNase I sensitivity of the two complementary nucleosomal DNA strands in cycloheximide-treated Ehrlich ascites tumor cells

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibited protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with 14C-thymidine and the newly synthesized strands - with bromodeoxyuridine and 3H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15-20% newly replicated DNA. Analysis of the distribution of 14C-labeled parental DNA fragments complementary to the 3H-nascent strand has shown that the 14C-labeled fragments prevail in the region of 30-50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5'-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems to be induced by interactions in the chromatin.