Nonreplicating DNA Research Articles

Replicative conformation of parental nucleosomes: salt sensitivity of deoxyribonucleic acid-histone interaction and alteration of histone H1 binding.

The transiently altered DNA-histone interaction of parental chromatin during replication was studied by micrococcal nuclease digestion. A large amount of nuclease-resistant pulse-labeled DNA and a small fraction of nonreplicating DNA are released from chromatin fragments by treatment with 0.5 M NaCl and appear as protein-free DNA. As shown by reconstitution experiments, the salt sensitivity of digested nascent chromatin is most probably a consequence of the shorter DNA fragment size (55 +/- 15 base pairs) in these complexes. This new DNA is associated with parental chromatin fragments which are structurally changed in such a way that parts of nucleosomal DNA were more susceptible to nuclease attack. The core histones of these particles are probably not distinct from those of salt-stable nucleosomes. However, histone H1 and probably high-mobility group proteins appear to be more weakly bound during replication as shown by electrophoresis under nondenaturing conditions. The results agree with the assumption that the transient alteration of nucleosomal conformation describes a state in which DNA could be replicated without leaving the associated core histone complexes. A possible attachment of pulse-labeled chromatin with nuclear matrix is discussed.

Characterization of DNA repair elicited by carcinogens and drugs in the hepatocyte primary culture/DNA repair test.

The autoradiographic unscheduled DNA synthesis measured in the hepatocyte primary culture/DNA repair test after exposure to chemical carcinogens was characterized. In order to document that this synthesis was occurring in nonreplicated DNA, density labeled, replicated DNA was separated from nonreplicated DNA by cesium chloride gradient centrifugation. Incorporation of [3H] thymidine into nonreplicated DNA was detected after exposure of hepatocytes to methyl methanesulfonate, an activation independent carcinogen, or N-2-acetylaminofluorene, an activation-dependent carcinogen, but not with fluorene, a noncarcinogen. N-4-Acetylaminofluorene, a mutagenic compound of uncertain carcinogenicity, also caused DNA repair. Both the autoradiographic assay and density gradient centrifugation detected incorporation of [3H] thymidine into DNA from the same hepatocyte preparation. By using density gradient centrifugation, DNA repair was detected in hepatocytes following exposure to the antihistamines pyrilamine maleate and tripelennamine HCl. Methapyrilene failed to elicit repair. Thus this study (1) verifies that the unscheduled DNA synthesis seen in the hepatocyte primary culture/DNA repair test after carcinogen exposure in DNA repair And (2) confirms the induction of DNA repair by pyrilamine maleate and tripelennamine HCl but not by methapyrilene.

The influence of hydroxyurea on the incorporation of exogenous DNA into host cells

The incorporation of exogenous isologous DNA into nuclei was studied in L-cells of replicating and non-replicating DNA. Hydroxyurea was used to inhibit DNA replication. The amount of exogenous DNA incorporated into cells decreased after the application of hydroxyurea by about a third. In the course of a 60-minute incubation exogenous DNA was incorporated into 75% of non-replicating DNA cells.

Dynamic association of replicating DNA fragments with the nuclear matrix of regenerating liver

Approx. 1–2% of the total nuclear DNA remains anchored to the proteinaceous nucleoskeletal matrix following endogenous DNA cleavage in isolated regenerating rat liver nuclei. The matrix-attached DNA fragments have an average size of 1.6 kilobases compared with 0.8 kilobases for total nuclear DNA, and are initially enriched with in vivo pulse-labeled, replicating DNA. Use of protease inhibitors during nuclear matrix isolation results in a several-fold enhancement of matrix-attached, newly replicating DNA. The percentage of replicating DNA which is anchored to the matrix rapidly declines from approx. 50% (one min pulse) to 5% after a 10 min pulse. The replicating DNA fragments are remarkably sensitive to release from the matrix structure by exogenous nucleases including pancreatic DNase I and micrococcal nuclease. Moreover, a preferential release of replicating as opposed to non-replicating DNA fragments is measured as long as substantial amounts of replicating DNA are matrix-attached. These findings suggest that the DNA replicational sites are continuously bound to the nuclear matrix. Two alternative mechanistic views termed the fixed and sliding matrix models are proposed to describe this association. In both models it is proposed that the DNA of the rat liver nucleus consists of ~ 125 000 matrix-attached DNA loops with an average size of 80 kilobases. The replication of DNA in replicon subunits then follows as a natural consequence of this inherent spatial organization.

Replication of SV40 chromatin in extracts from eggs of Xenopus laevis.

Simian virus 40 (SV40) nucleoprotein complexes were prepared from lytically infected cells and used as primer-templates for DNA replication in protein extracts from Xenopus eggs. We found that nucleoprotein containing replicating SV40 DNA served as primer-template while nucleoprotein with nonreplicating SV40 DNA was ineffective. In vitro DNA synthesis begins with short DNA fragments ("Okazaki fragments") which are, in later steps, joined to give unit length SV40 DNA strands, suggesting that in vivo initiated rounds of replication are completed in vitro in the Xenopus system. This conclusion is supported by a restriction enzyme analysis showing that in vitro DNA synthesis occurs in fragments distal to the SV40 origin of replication. Our studies indicate that SV40 DNA replication in Xenopus extracts can be used an an experimental system to study the biochemistry of replicative DNA chain elongation in vitro.

Site of Histone Assembly

Publisher Summary The chapter discusses the various site of histone assembly. DNA synthesis and histone synthesis are temporally coupled during the S phase of the cell cycle (1 -7). The doubling of the mass of DNA is matched by a doubling of histone protein. Since an obvious temporal deficiency in histone protein occurs at the replication fork, it is a reasonable assumption that newly synthesized histone protein is assembled into nucleosomes directly upon the vacancies on daughter-strand DNA. This seemingly straightforward problem has attracted the attention of numerous investigators. Although there is evidence that new histone protein is assembled on newly replicated DNA, other studies indicate that there is no preferential deposition on new DNA. The deposition (assembly) of newly synthesized histone protein into nucleosomes on newly replicated DNA versus nonreplicating DNA was investigated by methods employing the density-labeling of new DNA and by the nuclease sensitivity of new chromatin DNA relative to that of total chromatin. In all cases examined, no evidence was found favoring predominant deposition of new histones on new DNA. Rather, new histones appear to be assembled on both newly replicated DNA as well as on DNA that was not replicated during the experimental period.

Nucleotide sequence of Escherichia coli K-12 replication origin.

From subfragments of an EcoRI fragment (9 kilobase pairs) that contained the replication origin of the Escherichia coli chromosome and had been cloned as a recombinant with a nonreplicating DNA fragment coding for ampicillin resistance, small derivative plasmids were constructed. The smallest of these, pTSO151, contained a segment of 463 base pairs as the chromosomal component. Another plasmid, pSY134, constructed from BamHI digests of the EcoRI fragment and mini-F(pMF21), contained a region of 422 base pairs identical with a corresponding region in pTSO151. We conclude that the replication origin of E. coli chromosome is located within this 422-base-pair segment. The nucleotide sequence of this segment is presented.

DNA synthesis in toluene-treated bacteriophage-infected minicells of Bacillus subtilis

Bateriophage (Φ29, SPP1, or SPO1)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthesis as measured by incorporation of [ 3H]dTTP into a trichloroacetic acid-precipitable form. The [ 3H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2 · 10 5). Short exposure of the DNA molecules containing the incorporated [ 3H]dTMP to Escherichia coli exonuclease III results in over 90% of the [ 3H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, nalidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p- hydroxyphenylazo)-uracil .

Recently replicated simian virus 40 DNA is a preferential template for transcription and replication

The template activities for the processes of replication and transcription were compared for recently replicated ("new") and uniformly labeled ("old") simian virus 40 (SV40) DNA in infected monkey cells (line TC7). New SV40 DNA (pulse-labeled for 1 h) served as a template for a second round of replication with a relatively high probability (8% of the DNA replicated per h) for a period of 5 h, after which time its template activity rapidly decreased by severalfold. Old SV40 DNA (labeled for 24 h) functioned as a template for replication with a constant probability (1.8% of the DNA replicated per h) for at least 12 h. The proportion of RNA polymerase with nonreplicated and with recently replicated (bromodeoxyuridine-substituted) viral DNA was determined by an assay that used the Triton-soluble SV40 transcription complex. The proportion of RNA polymerase associated with nonreplicated SV40 DNA decreased very slowly (to 50% in 6 h), strongly suggesting that replicating viral genomes are not required as templates for the initiation of late transcription. This hypothesis was supported by the finding that the RNA synthesized in vitro was associated with covalently closed circular SV40 DNA. Furthermore, after 9 h in bromodeoxyuridine, the recently replicated viral DNA had nearly three times more RNA polymerase per unit of DNA than did the nonreplicated DNA. We thus conclude that recently replicated SV40 DNA is utilized preferentially as a template for transcription and for replication.

Distribution of DNA repair in chromatin

Nuclei from cells having the replicating DNA pulse-labeled with [3H]thymidine and the nonreplicating DNA uniformly labeled with [14C]thymidine were treated with micrococcal nuclease according to procedures which have been used to study the subunit structure of chromatin. Sedimentation analyses of chromatin from nuclease-treated nuclei, together with measurements of the size of newly synthesized DNA, indicate that (1) chromatin subunits near the replication fork are more susceptible to nuclease attack than subunits in non-replicating chromatin; (2) newly synthesized DNA is rapidly assembled into chromatin subunits prior to joining of small DNA replication intermediates; and (3) within 10 min after synthesis, DNA in newly replicated chromatin acquires a susceptibility to nuclease treatment similar to that of non-replicating chromatin.

Repair replication in replicating and non-replicating DNA after irradiation with UV light.

Ultraviolet light induces more pyrimidine dimers and more repair replication in DNA that replicates within 2 to 3 hr of irradiation than in DNA that does not replicate during this period. This difference may be due to special conformational changes in DNA and chromatin that might be associated with semiconservative DNA replication.

Segregation of Some Chromosomal Proteins during Cell Replication

The manner in which parental chromosomal proteins are distributed to daughter chromatin has recently been a matter of considerable interest. Jackson et al. (1975) followed the fate of radioactively labeled proteins and density-labeled DNA for several generations in rat hepatoma cells and concluded that histones are randomly distributed to the daughter DNA molecules. In further studies, they found that newly synthesized histone is not preferentially deposited on newly synthesized DNA (Jackson et al. 1976). Seale (1976a) noted a preferential deposition of pulse-labeled protein onto nonreplicating DNA. Micrococcal nuclease digestion studies, however, indicate that newly synthesized DNA associates with protein to form nucleosome spacings similar to those in parental chromatin (Seale 1976b; Weintraub 1976). These observations suggest that parental histones complex with newly synthesized DNA and align cooperatively along the double helix. Weintraub et al. (this volume) have shown that the nucleosome histones distribute conservatively as octamers, i.e., all histones in...

Cloning and mapping of the replication origin of Escherichia coli.

The replication origin of Escherichia coli has been cloned on a nonreplicating DNA fragment coding for ampicillin resistance. This recombinant DNA, named pSY211, replicates depending on the presence of the replication origin and can be recovered as a closed circular plasmid DNA of 10.7 megadaltons (Mdal). A restriction map has been constructed. EcoRI cleaves pSY211 into two fragments: one is the ampicillin fragment of 4.5 Mdal and the other is a chromosomal fragment of 6 Mdal and contains the origin. The 6 Mdal EcoRI fragment has four BamHI sites, three HindIII sites, and one Xho I site. A mutant of pSY211 has been isolated which is lacking two BamHI fragments of the chromosomal fragment. In recA hosts, pSY211 is lost at a high frequency. In recA+ hosts, pSY211 is integrated into the chromosome due to nucleotide sequence homology between pSY211 and the replication origin of the E. coli chromosome. The integration site has been mapped. We conclude that the replication origin is located at a site between uncA and rbsK, at about 83 min on the genetic map of E. coli.

Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli

Pulse-labeled ColEl DNA molecules, undergoing replication in Escherichia coli cells either in the absence or presence of chloramphenicol, were extracted and purified by neutral sucrose density gradient sedimentation and equilibrium centrifugation in an ethidium bromide-cesium chloride gradient. In the dye-buoyant density gradient, the replicating molecules were found in regions between the supercoiled and open-circular nonreplicating plasmid DNA, as well as in the open-circular region. In a neutral sucrose gradient, peaks of pulse label were found in the region of 26 to 38 S as well as at the 23 and 17 S positions corresponding to the positions of supercoiled and open-circular ColEl DNA. In alkaline sucrose gradient, nascent ColEl DNA was found to sediment as discrete peaks corresponding to 5-6, 7-9, and 14-16 S, indicating that at least one growing strand of the replicating molecule is produced discontinuously. In the electron microscope, many of the molecules appeared as partially supercoiled structures containing two open-circular branches of equal length, of less than 20% to more than 90% replicated. Branched open-circular molecules were not observed to any significant extent without prior treatment to induce single-strand scissions. The parental strands of the replicating molecules were determined to be covalently closed, but the superhelical density of the DNA was shown to be progressively decreased as replication proceeded.

Rapid assembly of newly synthesized DNA into chromatin subunits prior to joining of small DNA replication intermediates

Nuclei from cells having the replicating DNA pulse-labeled with [ 3H]thymidine and the nonreplicating DNA uniformly labeled with [ 14C]thymidine were treated with micrococcal nuclease according to procedures which have been used to study the subunit structure of chromatin. Sedimentation analyses of chromatin from nuclease-treated nuclei, together with measurements of the size of newly synthesized DNA, indicate that (1) chromatin subunits near the replication fork are more susceptible to nuclease attack than subunits in non-replicating chromatin; (2) newly synthesized DNA is rapidly assembled into chromatin subunits prior to joining of small DNA replication intermediates; and (3) within 10 min after synthesis, DNA in newly replicated chromatin acquires a susceptibility to nuclease treatment similar to that of non-replicating chromatin.

Association of nonreplicating P2 DNA to fast-sedimenting cell material following infection with satellite phage P4

Parental P2 DNA associates with some fast-sedimenting host material, probably the cell membrane, after infection of a sensitive cell. Membrane association is normal after infection of REP − cells, in which P2 DNA replication is blocked. Upon superinfection of a P2-lysogenic cell with an immunity-sensitive phage, association of the superinfecting P2 DNA does not occur. The A produce of P2 is required for membrane association. In a mixed infection of a P2 lysogen with P2 and its satellite phage P4, the superinfecting P2 DNA molecule does associate with the membrane. The P4-induced association does not require an active P2 A gene product. Some P4 gene activity is required, however, since uv-inactivated P4 is not able to induce it. In a P2 infection of a nonimmune cell the association of parental P2 DNA is an early event: it reaches 50% 8 min after addition of phage. The P4-induced association of P2 DNA after infection of P2-lysogenic cells does not reach 50% until 20 min after phage addition. The effect of P4 may be related to its ability to transactivate late P2 transcription in the absence of P2 A gene function.

Temporal relationships of chromatin protein synthesis, DNA synthesis, and assembly of deoxyribonucleoprotein.

Chromatin assembly has been investigated in terms of the sites on DNA where newly synthesized chromatin proteins associate. Chromatin from cells labeled with [14C]-BrdUrd and [3H]lysine was fixed with formaldehyde and resolved in CsCl gradients. By varying the spacing of the labeling intervals of the two isotopes so as to encompass all possible periods in S-phase, the association of labeled, newly synthesized proteins on newly synthesized (BrdUrd-substituted) or preexisting chromatin DNA was determined. In all experiments it was found that newly synthesized chromatin proteins predominantly associated with nonreplicating DNA. Possible mechanisms by which cells recycle preexisting chromatin proteins to restore the protein content of newly synthesized DNA are discussed.

Replication of adenovirus type 5 DNA in KB cells: localization and fate of parental DNA during replication.

Abstract The localization and fate of parental adenovirus type 5 (Ad5) DNA in KB cells at the time viral DNA replication occurs has been investigated by electron-microscope autoradiography and biochemical methods. At 18 hr after infection the majority of the parental Ad5 DNA molecules is present in the nucleus. About 60% of these molecules are involved in viral DNA replication by this time, but about 40% have not yet replicated at all. Electron-microscope autoradiography of cells infected with 3H-labeled Ad5 reveals that replicating parental Ad5 DNA molecules are randomly distributed throughout the nucleus, while nonreplicating viral DNA has a preference to locate in the periphery of the nucleus. Based on the experiments reported in this communication, the intranuclear fate of parental Ad5 DNA is discussed.

Covalently closed circular DNA molecules of low superhelix density as intermediate forms in plasmid replication.

COVALENTLY closed circular (CCC) duplex extrachromosomal DNAs are widespread in biological systems1. Because of their physical properties and small size, extra chromosomal circular DNAs are useful for studying the biology of genetic material. Their mechanism of replication is of particular interest and has been investigated extensively. Recent studies have shown that the replication of different types of circular DNA, such as plasmids of Escherichia coli, the DNA of polyoma and SV40 viruses, and mitochondrial DNA is basically similar2,3. Non-replicating circular DNA is isolated from cells largely as covalently closed supercoiled molecules (form I DNA). Because of its topological constraints, CCC DNA has a restricted affinity for intercalating dyes such as ethidium bromide and propidium diiodide; its buoyant density in gradients of CsCl containing such dyes is consequently much greater than that of non-circular or nicked circular (form II) DNA species, which can unwind to accommodate maximal amounts of dye4. DNA molecules in the process of replication have been shown to have buoyant densities intermediate to form I and form II DNA (refs 5–12 and L. Katz, P. H. Williams, S. Sato, R. W. Leavitt, and D. R. Helinski, unpublished). In a previous study of replicative forms of a constructed recombinant plasmid (pSC134) of E. coli13, we observed material pulse labelled with radioactive thymidine that had a buoyant density in CsCl–ethidium bromide gradients even greater than that of form I DNA5. Here we report results which suggest that this material, termed “heavy replicative DNA” or hrDNA, is a covalently closed circular DNA species that has a reduced number of supercoils, and is an intermediate in the replicative process.

New class of enzymes acting on damaged DNA.

ONE of the major pathways of DNA repair consists of the removal and replacement of damaged nucleotides in non-replicating DNA. In the classical excision repair model1,2, the first enzymatic step in this process is the introduction of a single-strand break in the DNA adjacent to a defective nucleotide residue. Endonucleases that specifically attack DNA containing pyrimidine dinners2 or apurinic sites3–5 have subsequently been isolated from many types of cells, and enzymes of these two types have been shown to be active in DNA repair in Escherichia coli by the isolation of repair-deficient mutant strains with defective endo-nucleases6,7.