The fate of parental nucleosomes during SV40 DNA replication.

The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.     

DNA synthesis in polyoma virus infection. IV. Mechanism of formation of closed-circular viral DNA deficient in superhelical turns.

A marked reduction in the rate of viral DNA synthesis is accompanied by an alteration to the superhelicity of progeny DNA in polyoma virus-infected cells in which protein synthesis has been inhibited by cycloheximide. Viral DNA molecules formed in the presence of cycloheximide consist predominantly of closed-circular monometric species (referred to as form Ic) characterized by a decreased superhelix density, corresponding to deltasigmao = 0.0195, as compared to form I DNA by propidium diiodide-cesium chloride isopycnic analysis. Form Ic is synthesized on pre-existing form I templates without the intervention of progeny form I as an intermediate. It is concluded that inhibition of protein synthesis results in the alteration of some process in the closure of daughter DNA that leads to a marked reduction of superhelical turns of progeny molecules. About two-thirds of form Ic molecules return to the form I conformation upon reversal of cycloheximide inhibition by a mechanism independent of DNA replication.     

phi X174-directed DNA and protein syntheses in infected minicells.

Phi X174-infected minicells, produced by Escherichia coli PC2251, synthesized 11 phi X174-encoded polypeptides. The infecting single-stranded viral genome was converted to a double-stranded, closed circular, replicative form (replicative form I). Little, if any, replicative form I replication took place, and synthesis of progeny single-stranded molecules could not be detected.     

Analysis of single-stranded DNA stability and damage-induced strand loss in mammalian cells using SV40-based shuttle vectors

The fate and stability of fully or partially single-stranded DNA molecules transfected into mammalian cells have been analysed. For this, we constructed a simian virus 40 (SV40)-based shuttle vector containing the f1 bacteriophage replication origin in the two possible orientations (pi SVF1-A and pi SVF1-B). This vector contains the SV40 origin of replication, the late viral genes and DNA sequences for replication and selection in Escherichia coli. It also carries the lacO sequence, which permits the analysis of plasmid stability. Single-stranded DNA from pi SVF1-A and pi SVF1-B were produced in bacteria and annealed in vitro to form a heteroduplex molecule. We showed that, in monkey kidney COS7 cells, single-stranded vectors replicate to form duplex molecules. After transfection of the three forms of molecules (single-stranded, heteroduplex or double-stranded), replicated DNA was rescued in E. coli. Vector stability was analysed by checking for plasmid rearrangements and screening for lacO mutants. The single-stranded pi SVF1 has a lower rearrangement level, while the spontaneous mutation frequency (on the lacO target) is in the same range as for the double-stranded vector. In contrast, the level of spontaneous mutagenesis is higher for the heteroduplex than for the single- and double-stranded forms. In addition, we found that replication of heteroduplex with one strand containing ultraviolet light-induced lesions yields progeny molecules in which the irradiated strand is mostly lost. This result indicates for the first time the specific loss of the damaged strand in mammalian cells.     

Intermediates in the replication of kinetoplast DNA minicircles

Kinetoplast DNA of Crithidia fasciculata and other trypanosomatids is in the form of a network of thousands of minicircles and a few dozen maxicircles. Minicircles replicate as free molecules after release from the network, and their progeny subsequently reattach to the network (Englund, P. T. (1979) J. Biol. Chem. 254, 4895-4900). The minicircles just released from the network are covalently closed and apparently completely relaxed. After Cairns-type (theta) replication, the two minicircle progeny have different structures. One has a nascent H (heavy) strand which initially is in the form of 20-110 nucleotide fragments that are separated by gaps (Kitchin, P. A., Klein, V. A., Fein, B. I., and Englund, P. T. (1984) J. Biol. Chem. 259, 15532-15539). The other initially has a full-size (2.5 kilobase) nascent L (light) strand. During the time between formation of these progeny molecules and network reattachment, the nascent L strand is nicked (or gapped) and nascent H strand is partially repaired. Therefore, both progeny, at the time of reattachment, have several nicks (or gaps) in their nascent strand. Minicircle progeny with a nascent L strand reattach to the network quickly, whereas those with a nascent H strand reattach more slowly. Once reattached to the network, the nicks or gaps in the minicircles are repaired until finally covalent closure occurs.     

Infectious linear DNA sequences replicating in simian virus 40-infected cells.

A new class of linear duplex DNA structures that contain simian virus 40 (SV40) DNA sequences and that are replicated during productive infection of cells with SV40 is described. These structures comprise up to 35% of the radioactively labeled DNA molecules that can be isolated by selective extraction. These molecules represent a unique size class corresponding to the length of an open SV40 DNA molecule (FO III), and they contain a heterogeneous population of DNA sequences either of host or of viral origin, as shown by restriction endonuclease analysis and nucleic acid hybridization. Part of the FO III DNA molecules contain viral-host DNA sequences covalently linked with each other. They start to replicate with the onset of SV40 superhelix replication 1 day after infection. Their rate of synthesis is most pronounced 3 days after infection when superhelix replication is already declining. Furthermore, they cannot be chased into other structures. At least a fraction of these molecules is infectious when administered together with DEAE-dextran to permissive cells. After intracellular circularization, superhelical DNA FO I with an aberrant cleavage pattern accumulates. In addition, tumor and viral capsid antigen are induced, and infectious viral progeny is obtained. Infection of cells with purified SV40 FO I DNA does not result in FO III DNA molecules in the infected cells or in the viral progeny. It is suggested, therefore, that these FO III DNA molecules are perpetuated within SV40 virus pools by encapsidation into pseudovirions.     

Mechanism of Replication of φX174 Single-Stranded DNA IX. Requirement for the Escherichia coli dnaG Protein

Escherichia coli NY73, possessing a temperature-sensitive mutation in the dnaG locus, was rendered sensitive to bacteriophage phiX174 by P1 transduction. phiX174 reproduces in this strain at 30 C but not at 40 C. All three stages of phiX174 replication, parental replicative form (RF) synthesis, RF replication, and progeny single-stranded DNA synthesis, are thermolabile in this mutant. Competition-annealing data show that both plus- and minus-strand synthesis are equally inhibited after shift up to 40 C during RF replication. We conclude that the dnaG gene product is required for the synthesis of both strands of phiX RF during RF replication and of the complementary strand and viral progeny strands during stages I and III, respectively.     

Escherichia coli topoisomerase I can segregate replicating pBR322 daughter DNA molecules in vitro

The reconstituted pBR322 DNA replication system has been used to identify a mechanism for the processing and segregation of daughter DNA molecules by Escherichia coli topoisomerase I (Topo I) during the terminal stages of DNA replication. At low concentrations of Topo I (sufficient to confer specificity to the replication system for DNA templates containing a ColE1-type origin of DNA replication), the major products of the replication reaction were: multigenome-length, linear, double-stranded DNA molecules (an aberrant product); multiply interlinked, catenated, supercoiled DNA dimers; and a last Cairns-type replication intermediate. Thirty- to fifty-fold higher concentrations of Topo I led to the appearance of form II and form I pBR322 DNA as the only synthetic products. A model was developed in which Topo I, bound to a single-stranded gap on the parental H strand DNA just upstream of the origin of DNA replication, catalyzed the decatenation of the intermolecular linkages between the two daughter DNA molecules that were generated by primosome-catalyzed unwinding of the residual nonreplicated parental duplex DNA in the last Cairns-type intermediate. At low concentrations of Topo I, however, the intermolecular linkages persisted and, within the context of this replication system, were not removed by DNA gyrase. In support of this model it was demonstrated that: there was a single-stranded gap between the nonreplicated parental duplex region and the 5' end of the nascent leading-strand DNA; the number of intermolecular linkages in the catenated supercoiled DNA dimers was inversely related to the concentration of Topo I; the supercoiled DNA dimers did not serve as a precursor of the final form I DNA product; and maturation of the last Cairns-type replication intermediate to form I DNA was not affected by the presence of coumermycin, a potent inhibitor of the activities of DNA gyrase.     

Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro

Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.     

Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).     

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.     

Telomere configuration influences the choice of telomere maintenance pathways

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In yeast cells that lack telomerase, telomeres are maintained by alternative type I and type II recombination mechanisms. Previous studies identified several proteins to control the choice between two types of recombinations. Here, we demonstrate that configuration of telomeres also plays a role to determine the fate of telomere replication in progeny. When diploid yeasts from mating equip with a specific type of telomeric structure in their genomes, they prefer to maintain this type of telomere replication in their descendants. While inherited telomere structure is easier to be utilized in progeny at the beginning stage, the telomeres in type I diploids can gradually switch to the type II cells in liquid culture. Importantly, the TLC1/tlc1 yeast cells develop type II survivors suggesting that haploid insufficiency of telomerase RNA component, which is similar to a type of dyskeratosis congenital in human. Altogether, our results suggest that both protein factors and substrate availability contribute to the choice among telomere replication pathways in yeast.     

Replication of pBR322 DNA in vitro with purified proteins. Requirement for topoisomerase I in the maintenance of template specificity

The replication of plasmid pBR322 DNA has been reconstituted with purified proteins from Escherichia coli. Initiation of the leading-strand requires RNA polymerase holoenzyme, DNA polymerase I, RNase H, and DNA gyrase. Initiation of the lagging-strand requires the primosomal proteins (the dnaB, dnaC, and dnaG proteins, replication factor Y (protein n') and proteins i, n, and n") and the single-stranded DNA binding protein. DNA polymerase III holoenzyme is required for extensive elongation of the nascent DNA chains. The products of this replication reaction are primarily nonsegregated daughter molecules. However, the addition of small amounts of soluble extract from E. coli results in the completion and segregation of these molecules to give mature form I DNA, suggesting that additional factors are required for this process. Topoisomerase I is necessary to make the replication system specific for pBR322 DNA as a template, indicating that the linking number of the DNA, determined by an equilibrium between the opposing activities of topoisomerase I and DNA gyrase, plays a crucial role in determining the reactivity of the DNA molecule toward initiating DNA replication. The function of the proteins involved in the replication of this closed-circular, double-stranded, superhelical DNA is discussed.     

Synthesis of multimeric polyoma virus DNA in mouse L-cells: role of the tsA1S9 gene product.

Several different forms of progeny viral DNA can be identified in polyoma virus (Py)-infected mouse L-cells. The majority comprise mature form I superhelical DNA and the circular, double-stranded "theta" replicating intermediates in which the progeny DNA strands never exceed the unit genome length of the template. There is formed, in addition, a minority fraction of multimeric, linear, double-stranded Py DNA molecules that sediment heterogeneously at 28 to 35S and greater than 35S. Restriction enzyme analysis of these large Py DNA molecules reveals them to be tandem arrays of multiple unit genome lengths, covalently linked head to tail. It is estimated that the 28 to 35S multimeric DNA has an average size of about 20 megadaltons, made up of 6 to 20 Py genome units. The greater than 35S Py DNA is, of course, larger. Kinetic analysis indicates that formation of the monomeric progeny viral DNA and the 28 to 35S multimeric Py DNA reaches a peak at about 35 to 36 h postinfection. Synthesis of the very large linear molecules of greater than 35S is first detected after this interval and continues thereafter. The de novo synthesis of all of these progeny Py DNA molecules proceeds apparently normally in Py-infected tsA1S9 mouse L-cells incubated at 38.5 degrees C under conditions which restrict normal cellular DNA replication. These findings suggest that the cellular DNA topoisomerase II activity, encoded in the tsA1S9 locus (R. W. Colwill and R. Sheinin, submitted for publication), is not required for de novo formation of any form of Py DNA. However, the total amount made and the rate of synthesis of the large molecular weight Py DNA are affected very late in temperature-inactivated tsA1S9 cells.     

Serum lipid profiles of young Japanese women with iron deficiency without anemia

PurposeWe aimed to evaluate the association between iron deficiency without anemia (IDNA) and serum lipid profiles in young women of around 20 years of age.MethodsThis study included non-anemic (hemoglobin ≥ 12 g/dL) female volunteers aged 18 to 22 years who were not taking mineral/vitamin supplements and living in the metropolitan area of Tokyo, Japan. These volunteers were classified into two groups based on their sFer (serum ferritin) levels: normal group (sFer ≥ 20 ng/mL, n = 36) and IDNA group (sFer < 20 ng/mL, n = 29). Venous blood samples were obtained from the antecubital veins of these volunteers after 10–12-h fasting to measure the hematological and biochemical parameters, including lipid levels and iron status. The results of each group were compared using Student’s t-test or the Mann–Whitney U test (for inhomogeneous variance).ResultsThe serum cholesterol levels varied depending on the iron status in the women. Serum high-density lipoprotein cholesterol (HDL-C) levels in the IDNA group were significantly higher (P = 0.006) than that in the normal group. However, the levels of total cholesterol (T-CHO), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were not significantly different between the groups. Serum LDL-C levels were positively and significantly correlated with sFer levels in the IDNA group (Kendall’s rank correlation 0.264, P = 0.044), but not in the normal group. The sFer level was not correlated with serum HDL-C in both groups. The reason for the high serum HDL-C levels in young women with IDNA is not yet clear. Compared to the normal group, the frequency of eating bread containing bran was significantly higher (P = 0.031) and that for yogurt was significantly lower (P = 0.040) in the IDNA group. The proportion of the women who were susceptible to infection, which was measured using the Cornell Medical Index, was significantly higher in the IDNA group than in the normal group. Among those susceptible to infection, the serum HDL-C level of the volunteers in the IDNA group was significantly higher than that of the volunteers in the normal group (P = 0.024).ConclusionsOur findings suggest that lipid parameters may be associated with IDNA and susceptibility to infection. Further research is needed to elucidate the mechanisms underlying the changes in the serum cholesterol levels in individuals with IDNA and the clinical significance of these findings.     

Binding and uptake of Col 1(I), a peptide capable of inhibiting collagen synthesis in fibroblasts

Col 1(I), a collagenase-resistant segment of the amino-terminal propeptide of pro alpha 1(I) chains, is known to inhibit collagen synthesis in cultured skin fibroblasts and also in a cell-free protein synthesizing system by reducing the translation of procollagen mRNA. These findings prompted us to explore the fate of exogenous Col 1(I) in the cellular processing of human skin fibroblasts using colloidal gold labeled protein (Col 1(I)-Au). Distribution of Col 1(I)-Au on the cell surface was studied by the platinum-carbon replication technique. Three different types of binding pattern could be observed: 1) Binding sites in the form of a fibrillar network, 2) those in the form of clusters, and 3) solitary bound gold conjugates. The latter two cases were determined to be specific. The intracellular routing of Col 1(I)-Au was studied by thin sections. Specifically bound gold conjugates were found in coated pits and after the initiation of the internalization process in coated vesicles and endosomes. Acid phosphatase cytochemistry revealed that only a small amount of Col 1(I)-Au is delivered to lysosomes. The bulk of gold conjugates is present even after prolonged incubation at 37 degrees C in acid phosphatase-negative compartments of the cell. Our data suggest a mechanism in which Col 1(I) initially is bound to the cell surface and subsequently internalized via the coated pit-coated vesicle pathway.     

Multiple and Specific Initiation of T4 DNA Replication

Partially replicated T4 DNA molecules (PRM) whose parental or progeny DNA was labeled with bromodeoxyuridine BUdR was analyzed by gradual shearing followed by CsCl banding of the sheared product. Analysis of PRM containing 18-mum replicated DNA showed that each replicated region was 3- to 6-mum long, indicating three to 6 replicative sites per molecule. Analysis of PRM containing 9-mum replicated DNA similarly indicated two to three replicated regions per molecule. DNA from the replicated regions of PRM containing 10-mum replicated DNA ("donor") was hybridized to DNA from mature phage ("recipient"), and the resulting hybrid was subjected to digestion with exonuclease I. The extent of protection of the recipient and more efficient self-annealing of progeny fragments from PRM indicated that the replicated regions represented 8 to 10 nonrandom locations of the genome. Possible significance of multiple sites for initiation of DNA replication is discussed.