DNA synthesis associated with a DNA-nuclear-membrane complex from rat liver

Summary Ultrastructural analysis of M-band from nuclei of rat liver showed small amounts of chromatin, fragments of inner nuclear membrane, some amorphous nuclear material, and nucleopores. The outer nuclear membrane with its associated ribosomes was removed by Sarkosyl during the preparation of the M-band sample. Morphological features of nucleopores and the inner nuclear membrane were confirmed by freeze-fracture technique. The gross chemical composition of the M-band was similar to that of nuclear membrane fractions prepared by other techniques. The M-band contained the greatest proportion of newly-labeled DNA and also supported DNA synthesis in vitro. Electron-microscopic autoradiography of the M-band showed localization of silver grains of thymidine-³H presumably over newly synthesized DNA. The DNA synthesis could not be attributed to spurious attachment of DNA polymerase to M-band during its isolation. It was partially removed from the M-band by treatment with 0.5 M KC1, phospholipase A or C; and completely, by the action of pancreatic DNase. DNA synthesis was greater in M-band fractions isolated from nuclei of 24-hour regenerating liver.Supported in part by a grant (CA 10298) from the National Cancer Institute     

The anchoring of newly synthesized adenovirus DNA to the nuclear matrix

After adenovirus infected HeLa cells were pulse labeled and pulse-chase labeled with 3H-thymidine, the nuclear matrix and DNA remaining tightly bound to the matrix were obtained by sequential cell fractionation. Measuring the radioactivity of labeled DNA indicated that newly synthesized viral DNA specifically attach to the nuclear matrix and the amount of binding DNA is in direct proportion to the viral DNA replication activity: then the DNA gradually detach from the matrix and are involved in viral assembly. Electron microscopic autoradiography of the extracted cells showed the virion and viral DNA associated with the nuclear matrix, and thus further confirmed the anchoring of newly synthesized viral DNA to the nuclear matrix.     

Ultrastructural Studies of H-1 Parvovirus Replication VI. Simultaneous Autoradiographic and Immunochemical Intranuclear Localization of Viral DNA Synthesis and Protein Accumulation

The localization of H-1 viral replicative-form double-stranded DNA and progeny single-stranded DNA replication in parasynchronously infected, simian virus 40-transformed newborn human kidney cells was studied with high-resolution electron microscope autoradiography (80-nm silver grains). We analyzed wild-type H-1 and ts1 H-1 (a conditional mutant defective in progeny single-stranded DNA synthesis). The proportion of the total DNA synthesis that was viral was estimated to be >90% by comparing the amount of [(3)H]thymidine uptake in cultures infected with wild-type H-1 versus ts14 (an H-1 mutant defective in DNA replication). Simultaneous staining with cytochrome c-conjugated anti-H-1 immunoglobulin G was performed to ensure that cells incorporating [(3)H]thymidine (2- to 60-min pulses) were H-1 infected. The sites of H-1 replicative-form (in ts1-infected cells) and progeny (in wild-type-infected cells) DNA synthesis were identical. Immunospecifically labeled nuclei at the earliest stages of infection exhibited dense clusters of silver grains over material extruded from nucleolar fibrillar centers. These foci became larger with increasing cellular damage, forming a limited number of H-1 DNA synthetic centers in the euchromatin. Each island-like focus was surrounded by tufts of heterochromatin containing high concentrations of unassembled H-1 capsid proteins. In late phases of infection, the heterochromatin became completely marginated, and the nucleoplasm contained only euchromatin that exhibited randomly distributed sites of H-1 DNA replication. This indicates that H-1 DNA synthesis begins at localized euchromatic or nucleolar sites and then spreads outward. Immunostained heterochromatin and nucleolar chromatin never incorporated [(3)H]thymidine. Our results suggest that H-1 proteins and cellular cofactors associated with the fibrillar component of the nucleolus and the euchromatin may play a role in the regulation of H-1 DNA synthesis.     

Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS- polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000- 75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.     

DNA POLYMERASES IN FRACTIONATED RAT BRAIN NUCLEI

Abstract— —Adult rat brain nuclei were separated by discontinuous sucrose gradient centrifugation into astrocyte enriched, neuron enriched, and oligodendrocyte/microglia fractions. Nuclear fractions were subjected to velocity sucrose gradient centrifugation and gradient fractions assayed using relatively specific reaction mixtures for DNA polymerase-α, -β and TdT. NEM resistant DNA polymerase activity (DNA polymerase-β) was detected in equivalent amounts in all nuclear fractions. High molecular weight NEM sensitive activity (DNA polymerase-α) was found primarily in the neuron enriched fraction. The significance of the presence of DNA polymerase-α, an enzyme thought to be involved in DNA replication, in a cell incapable of cell division is unknown. TdT was detected in all fractions with increased activity in the neuron enriched fraction. The finding of TdT in thymocytes and neurons further supports the hypothesis that this enzyme is involved in the storage of noninherited information.     

Adenovirus DNA synthesis in vitro in an isolated complex.

DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.     

Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication.     

DNA replication and the nuclear membrane

To investigate the relationship between the nuclear membrane and DNA replication, Chinese hamster cells were labeled with tritiated thymidine and examined by electron microscope autoradiography. Unsynchronized cells were labeled for periods ranging from 0.5 to 20 minutes. There was no relative increase in the frequency of membrane-associated grains with the shorter labeling times, indicating that the replication point is not necessarily close to the nuclear membrane. When cells were synchronized to the beginning of the S period with mitotic selection and hydroxyurea, the percentage of membrane-associated grains was very low, indicating that DNA synthesis is not initiated at the nuclear membrane. When cells synchronized by mitotic selection were labeled at various times throughout the cell cycle, the percentage of peripheral grains was low in early S period and became progressively higher toward late S period as heterochromatin began to replicate. The labeling of Unsynchronized Microtus agrestis cells indicated that much of the peripheral labeling is due to the replication of intercallary heterochromatin. The results indicate that there is no association between the nuclear membrane and DNA replication.     

Synthesis of the adenovirus-coded DNA binding protein in infected cells.

Synthesis of the 75K (75K indicates a moleculatr weight of 70,000 to 75,000) DNA binding protein, an early virus-coded protein in adenovirus 2-infected KB cells, and its regulation were studied by using a radioimmune precipitation inhibition assay. The protein was first detected at 4 h postinfection and accumulated at an expoential rate. An arrest of further synthesis (accumulation) was observed at 10 to 11 h postinfection, coinciding with the onset of synthesis of late virion proteins. In contrast, when the infected cells were treated with 25 mug of arabinosyl cytosine per ml to block viral DNA replication, the synthesis of 75K protein did not cease but continue for up to 36 h postinfection. The synthesis of 75K protein in cells after release from a cycloheximide block (2 to 9 h postinfection) was analyzed. Increased amounts of early adenovirus-specific mRNA accumulate in infected cells during a cycloheximide block (Parsons and Green, 1971). However, cycloheximide treatment did not produce increased levels of 75K protein, and an abrupt arrest of 75K protein formation was again observed at the time of synthesis of late virion proteins. Partition of the 75K protein between the nuclear and cytoplasmic fractions during the course of infection was studied. The 75K protein appeared first in the cytoplasm and then in the nucleus after a slight lag. Accumulation of the 75K protein continued both in the cytoplasm and nucleus, with higher levels being found in the cytoplasm.     

Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.     

T-antigen interactions with chromatin and p53 during the cell cycle in extracts from xenopus eggs.

The role of SV40 large tumor T-antigen in replication of viral DNA is well established, but it is still unclear how T-antigen triggers cellular replication and cell transformation in non-permissive cells. Here, we used Xenopus egg extracts which reproduce most nuclear events linked to the cell cycle in vitro to analyze its interaction with genomic chromatin during the cell cycle. We show that T-antigen associates with chromatin before the nuclear membrane formation, and further demonstrate that the nuclear membrane is not necessary for its import into the nucleus. We show that the interaction of T-antigen with the endogenous chromatin does not occur at replication foci nor at RPA pre-replication centers. Immunoprecipitations as well as sucrose gradient experiments, indicate that the endogenous pool of p53 interacts with T-antigen. In addition, a transient association of both proteins with the nuclear matrix is observed during the ongoing DNA synthesis. These data are discussed in view of the T-antigen and p53 activity during the cell cycle.     

T4 DNA replication and viral gene expression

The normal dependence of “late” T4 gene expression on concurrent viral DNA replication is circumvented in cells infected with a triple mutant in which viral DNA polymerase, DNA ligase, and the exonuclease functions of genes 46 or 47 are defective. Acrylamide gel electrophoresis of labeled proteins from infected cells has made possible an extension of the analysis of replication-uncoupled T4 protein synthesis. We find a number of late T4 proteins synthesized: the products of genes 34, 37, 18, 23 and 24. Processing of the gene 23 product, normally headassembly dependent, occurs, but with considerably diminished efficiency compared to wild-type infection. Late T4 protein synthesis in replication-uncoupled infection retains a requirement for T4 gene 33 and gene 55 function. Finally, a number of “early” T4 gene products, normally shut off late in wildtype infection, continue to be synthesized late in replication-uncoupled infection, concurrently with the late proteins.     

Rifampin and vaccinia DNA.

The effect of rifampin on the replication of vaccinia DNA was studied in mouse L cells by a cytochemical techinque and by alkaline sucrose sedimentation analysis of newly synthesized viral DNA molecules. By the use of a fluorescent DNA-binding compound (Hoechst 33258), the sequential appearance, size, and location of the viral "factories" in rifampin-treated, virus-infected cells were found to be indistinguishable from those observed in untreated, infected cells. Sedimentation analysis in alkaline scurose gradients of the viral DNA molecules labeled in pulse-chase experiments showed that formation of small fragments, elongation into "intermediate"-sized molecules, and maturation into full-length viral DNA and, finally, into cross-linked viral DNA molecules occurred in the absence or presence of rifampin. The results support the view that the primary effect of the drug is related to assembly or morphogenesis.     

Mechanisms and age-specific characteristics of nuclear DNA replication

Replication mechanisms of nuclear DNA in eucaryotic cells and their changes with the organism aging are discussed. Tge polyrepliconic nature of replicating DNA, discontinuous synthesis of its newly formed chains, enzymic apparatus of replication and regulation of this process are described. Special attention is paid to destabilization of the secondary structure of the replicating molecule. In aging, changes in the DNA replication were found at each level of its regulation, i.e. at synthesis precursors, enzymic replication complex and replicating DNA structure. This may be an indication of the altered regulation of the DNA replication with aging.     

Adenovirus DNA synthesized in the presence of aphidicolin

Adenovirus types 2 and 5 DNA synthesized in vivo and in vitro in the presence of aphidicolin were studied. Inhibition of adenoviral DNA synthesis by aphidicolin was only 70% even at a concentration of 30 micrograms/ml of aphidicolin, at which the cellular DNA synthesis was completely inhibited. When initiation of the viral DNA synthesis was synchronized with hydroxyurea and labeled with [3H]thymidine for 60 min, the viral DNA synthesized in the presence of 30 micrograms/ml of aphidicolin was not of full length (35 kb) but small (approximately 12 kb) by analysis of alkaline sucrose density gradient centrifugation. When initiation of the viral DNA synthesis was not synchronized, the viral DNAs ranging from full size to 12 kb were synthesized in the presence of aphidicolin, indicating that the nascent DNAs longer than about 12 kb can continue to elongate in the presence of aphidicolin. This 12 kb DNA was not derived from the degradation products of newly synthesized full size adenoviral DNA. The viral DNA synthesis was restored and the full size of adenoviral DNA was attained within 15 min following removal of aphidicolin. About 20% of the entire viral genome length from the 5'-end was not inhibited by aphidicolin, while the synthesis of interior fragments of the adenoviral DNA was markedly inhibited by aphidicolin, judging from the electrophoretic pattern on neutral agarose gel after digestion of DNA with Hind III. These results indicate that aphidicolin inhibits adenoviral DNA replication at the internal region located approximately 20-30% from both terminals.     

Fractionation and characterization of DNA at sites of replication from rat liver nuclei

Regenerating rat liver nuclei when sonicated and centrifuged in a Cs2SO4 gradient were fractionated into three distinct bands. These bands were designated as light band (LB), middle band (MB), and heavy band (HB) according to their density. LB and MB were shown to consist of large granular particles with varying electron densities, but LB also contained remnants of nuclear membrane. When analysed by gel electrophoresis, LB and MB displayed more than 35 bands of proteins. The third fraction, HB, consisted largely of small chromatin fibers and its proteins were predominantly the four histones of the nucleosomal core particle. Following short pulses with [3H]thymidine in vivo, the specific activity of DNA in LB and MB was significantly higher than that of bulk DNA contained in HB. DNA in all three fractions became equally labelled as the duration of the labelling interval increased beyond 30 min. Newly synthesized DNA was characterized by electrophoresis on analytical 1.7% acrylamide -0.5% agarose composite gels. After a 1-min labelling interval in vivo, 17% of the rapidly labelled DNA from LB and MB was stationary at the gel origin like replication forks from E. coli, but only 3% of HB DNA had zero mobility. Electron microscopy confirmed the presence of DNA replication forks in LB, MB, and HB. With increasing time of synthesis the proportion of labelled DNA exhibiting zero mobility decreased in all three fractions. Denaturation of DNA or digestion of single-stranded DNA with S1 nuclease released newly synthesized DNA from the gel origin. Ribonuclease was without effect. DNA recovered from LB and MB also had a higher molecular weight than the HB DNA. Together these results indicate (1) that LB and MB are enriched in newly replicated DNA; (2) that an increased proportion of newly replicated DNA in LB and MB is associated with DNA replication forks; and (3) that the replicating DNA recovered in LB and MB may be associated with other nuclear constituents in situ because this DNA appears to be protected from the more frequent chain breaks introduced into the bulk of chromatin (HB) by sonication.