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PubMed1976

DNA replication and post-replication repair in U.V.-sensitive mouse neuroblastoma cells.

Mouse neuroblastoma cells differentiate when grown in the absence of serum; differentiation is reversed on the addition of serum. Differentiated cells are more sensitive to U.V.-radiation than proliferating cells. Whereas addition of serum to differentiated neuroblastoma cells normally results in immediate, synchronous entry into S phase, irradiation just before the addition of serum results in a long delay in the onset of DNA replication. During this lag period, incorporated 3H-thymidine appears in the light density region of CsCl gradientss, reflecting either repair synthesis or abortive replication. Post-replication repair (gap-filling) was found to be present in proliferating cells and at certain times in differentiated cells. It is suggested that the sensitivity of differentiated neuroblastoma cells to U.V.-radiation may be due to ineffective post-replication repair or to deficiencies in more than one repair mechanism, with reduction in repair capacity beyond a critical threshold.

Cell Differentiation↗
PubMed1990

Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2) can coinfect and simultaneously replicate in the same human CD4+ cell: effect of coinfection on infectious HSV-2 and HIV-1 replication.

Experiments were designed to determine whether HIV-1 and herpes simplex virus type 2 (HSV-2) coinfection leads to simultaneous replication of both viruses in the same human CD4+ cell (MT-4 cell line) and the possible effects of coinfection on infectious virus production. Results from transmission electron microscopy analysis revealed replication of typical HSV-2 nucleocapsids in the nucleus and budding of HIV-1 particles through the plasma membrane and through intracytoplasmic vacuoles containing enveloped HSV-2 particles in the same coinfected cell. Coinfection of HIV-1 persistently infected H9IIIB or promonocytic U1 cells with HSV-2 did not alter total production of infectious HSV-2 or the percentage of HSV-2 infectious centers compared with control H9 and U937 cells infected with HSV-2 alone. However, in coinfected promonocytic U1 cells HSV-2 induced infectious HIV-1 production measured by syncytial plaque assay. In summary, both HIV-1 and HSV-2 can coinfect and simultaneously replicate in the same human CD4+ cell. Interactions between HIV-1 and HSV-2 appear to be unidirectional, resulting in accelerated replication of HIV-1 as reported by Albrecht et al. (J Virol 1989;63:1861-1868), but not HSV-2 as shown by us.

Acquired Immunodeficiency Syndrome↗
PubMed1990

SOS-dependent replication past a single trans-syn T-T cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells.

We have transfected SOS-induced and uninduced cells of a uvrA6 strain of Escherichia coli with single-stranded M13mp7-based vectors that carried a single trans-syn T-T cyclobutane dimer at a unique site. Unlike constructs carrying the cis-syn isomer of this lesion, these vectors could be replicated with modest efficiency (14%) in the absence of SOS induction and therefore provided an opportunity to measure directly the influence of such induction on error rate and mutation spectrum. We found that translesion synthesis in the absence of SOS induction was remarkably accurate; only 4% of the replicated bacteriophage contained mutations, which were exclusively targeted single T deletions. In SOS-induced cells, error frequency increased to 11% and the resulting mutations included targeted substitutions and near-targeted single base additions, as well as the T deletions. Replication efficiency was 29% in these conditions. SOS induction therefore leads not only to an enhanced capacity to replicate damaged DNA but also to a marked change in mutation frequency and spectrum.

Base Sequence↗
PubMed1976

Replication of T4 DNA in vitro. II. Assay system for and some properties of gene products required for T4 DNA replication.

[3H]dTTP was not incorporated into T4 DNA in the in vitro system for T4 DNA replication when the system was prepared from cells infected with T4 amber mutants defective in DNA replication. [3H]dTTP incorporation was resumed by adding the missing gene product to the defective system. DNA replication by the reconstituted system proceeded by the discontinuous mode of replication, as observed in the wild-type system. By using this in vitro complementation system, molecular weights of gene 41, 43, 44, 45, and 62 products in the active form were roughly estimated as 60,000, 130,000, 130,000, 60,000, and 130,000, respectively. Complex formation between the products of genes 44 and 62 was detected. Other strong interactions between the gene products tested were not detected by glycerol density gradient sedimentation. Interaction of gene products with denatured DNA was analyzed by using a DNA-agarose column, and the results showed that products of genes 32 and 43 had a strong affinity for DNA.

Coliphages↗
PubMed1991

Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2.

The TrfA proteins, encoded by the broad host range plasmid RK2, are required for replication of this plasmid in a variety of Gram-negative bacteria. Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame. The two proteins have been purified from Escherichia coli to near homogeneity as a mixture of wild-type TrfA-44/33, as TrfA-33 alone and as a functional variant form of TrfA-44, designated TrfA-44(98L), which contains a leucine in place of the TrfA-33 methionine start codon. Cross-linking experiments demonstrated that TrfA-33 can multimerize in solution. By using gel mobility shift and DNase I footprinting techniques the binding properties of TrfA-33, TrfA-44(98L), and TrfA-44/33 to the origin of replication of plasmid RK2 were analyzed. All three protein preparations were able to bind very specifically to the cluster of five direct repeats (iterons) contained in the minimal origin of replication. Each protein preparation produced a ladder of TrfA/minimal oriV complexes of decreasing electrophoretic mobility. The DNase I protection pattern on the five iterons was identical for all three protein preparations and extended from the beginning of the first iteron to 5 base pairs upstream of the fifth iteron. Studies on the affinity of the proteins for DNA fragments containing one, two, or all five iterons of the origin revealed a strong preference of TrfA protein for DNA containing at least two iterons. To study the stability of TrfA.DNA complexes, association and dissociation rates of TrfA-33 and DNA fragments with one, two, or five iterons were measured. This analysis showed that unlike complexes involving two or five iterons the TrfA/one iteron complexes were highly unstable, suggesting some form of cooperativity between proteins or iterons in the formation of stable complexes and/or the requirement of specific sequences bordering the iterons at the RK2 origin of replication for the stabilization of TrfA/DNA complexes.

Bacterial Proteins↗
PubMed1990

[Replication characteristics of plasmids and their dependence of recA gene for the initiation of Escherichia coli chromosome replication in the integrated state].

E. coli dnaA46 fails to grow and form colonies at 40 degrees C; integrated with plasmid enables it to grow at 40 degrees C or 42 degrees C. In a previous paper we reported that the replication of chromosome initiated by the integrated F' plasmid was recA gene dependent. In this paper we report further the behavior of 5 plasmids and 2 phages as to their dependence of recA gene in the integrated state. It was demonstrated that the dependence or independence of recA gene is not related to the direction of replication of the plasmids or phages in the free state. The hypothesis that the function of recA gene is to convert the unidirectionally replicating machinery in the free state to the bidirectionally replicating one in the integrated state is refuted accordingly.

Chromosomes, Bacterial↗
PubMed1992

DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells.

Genomic methylation patterns are established during maturation of primordial germ cells and during gametogenesis. While methylation is linked to DNA replication in somatic cells, active de novo methylation and demethylation occur in post-replicative spermatocytes during meiotic prophase (1). We have examined differentiating male germ cells for alternative forms of DNA (cytosine-5)-methyltransferase (DNA MTase) and have found a 6.2 kb DNA MTase mRNA that is present in appreciable quantities only in testis; in post-replicative pachytene spermatocytes it is the predominant form of DNA MTase mRNA. The 5.2 kb DNA MTase mRNA, characteristic of all somatic cells, was detected in isolated type A and B spermatogonia and haploid round spermatids. Immunobolt analysis detected a protein in spermatogenic cells with a relative mass of 180,000-200,000, which is close to the known size of the somatic form of mammalian DNA MTase. The demonstration of the differential developmental expression of DNA MTase in male germ cells argues for a role for testicular DNA methylation events, not only during replication in premeiotic cells, but also during meiotic prophase and postmeiotic development.

Animals↗
PubMed1978

New late gene, dar, involved in the replication of bacteriophage T4 DNA. III. DNA replicative intermediates of T4 dar and a gene 59 mutant suppressed by dar.

A mutation in the dar gene of phage T4 restored the arrested DNA synthesis caused by the gene 59 mutation. We have studied the DNA replicative intermediates in cells infected with a dar mutant and a dar-amC5 (gene 59) mutant by velocity sedimentation in neutral and alkaline sucrose gradients. In T4 dar-infected cells, compared to the wild type, three kinds of abnormalities were observed in DNA replication (i) There were unusually rapidly sedimenting intermediates (800S). (ii) When centrifuged in alkaline gradients, there was less single-stranded DNA exceeding 1 phage unit. (iii) The rate of repair of DNA intermediates was slower. It has been proposed by others that the 200S DNA replicative intermediates are required for DNA packaging, but our results showed that the 800S DNA of dar does not have to be converted into the 200S form to undergo conversion to mature viral DNA. Therefore, 200S DNA may not be an obligatory intermediate for mature viral DNA formation. In amC5 (gene 59)-infected cells, the DNA was completely converted 2 to 3 min after intiation of replication to the biologically inactive 63S DNA, and DNA synthesis was concomitantly arrested. However, in dar-am-C5 (gene 59)-infected cells, the formation of abnormal 63S DNA did not occur and 200S DNA appeared instead. An endonucleolytic activity, normally associated with the cell membrane and capable of making double-stranded cuts, was found in the cytoplasm of T4 dar-infected cells. Because the total activity of this endonuclease is the same for both wild-type T4D and the dar mutant, it seems unlikely that the dar protein has endonucleolytic activity itself. However, the finding does explain the abnormal sedimentation of dar DNA intermediates (800S) as well as the proposed suppression mechanism of the gene 59 mutation.

Centrifugation, Density Gradient↗
PubMed1979

Replication and maturation of phage P22 in a mutant of Salmonella typhimurium temperature sensitive in initiation of DNA replication.

TB37 is a dna A-mutant of Salmonella typhimurium in which the initiation of DNA replication at the origin is stopped at 42 degrees C. DNA synthesis in uninfected cells of this strain and in cells infected by phage P22 was followed by the pulse labelling technique. DNA replication ceases completely after about 50 minutes at the high temperature. After lytic infection with P22 (c2) at this time, DNA synthesis starts immediately and increases at a rate well comparable to the permissive control. Obviously the temperature sensitive function of the dnaA-product is dispensable for P22 DNA replication, especially for its initiation. This result is confirmed by the normal yield of phage particles under these conditions, provided that a late step in P22 maturation which naturally is temperature sensitive can proceed at low temperature. If TB37 is infected at 42 degrees C with P22 wild type, an unexpected high rate of phage controlled DNA synthesis is observed. Preliminary results seem to indicate that the process of integration is a prerequisite for part of this synthesis.

DNA Replication↗
PubMed1990

The cytoplasmic HBVDNA implication: HBV replication or non-replicative status.

Intrahepatocellular HBVDNA was investigated in patients with chronic hepatitis B by in situ hybridization assay, employing biotin-labelled HBVDNA as the specific probe. It was found that HBVDNA was predominantly present in the cytoplasm of hepatocytes. In general the cases with cytoplasmic HBVDNA could be grouped as follows: 1) cytoplasmic HBVDNA coinciding with intrahepatic envelope protein and nucleocapsid component expression, this pattern of the cytoplasmic HBVDNA would be replicative; 2) cytoplasmic HBVDNA accompanied by intrahepatic single HBsAg expression, common to HBVDNA integrated into host genome with only HBsAg being found frequently in liver; 3) with only cytoplasmic HBVDNA, with neither HBsAg nor HBcAg expression, representative for non-replicative status. It is speculated that the reason for the occurrence of the cytoplasmic hybridization signal in cases with non-replicative HBVDNA may be due to DNA-RNA transcript hybrid. The prudent way to evaluate the implication of the cytoplasmic HBVDNA is to consider the combination of HBVDNA location, intrahepatic gene product expression, and HBV markers in the serum.

Cytoplasm↗
PubMed1991

Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication.

Replication protein A (RP-A) is a three-subunit single-stranded DNA-binding protein that has been isolated from human cells. RP-A is essential for SV40 DNA replication and may also be important in genetic recombination. The sequence of a cDNA encoding the 70-kDa subunit of human RP-A is reported. The 616-amino acid predicted open reading frame of the human protein is 31% identical with the 621-amino acid open reading frame of the 70-kDa subunit of RP-A from the yeast Saccharomyces cerevisiae. Both proteins share a highly conserved putative metal binding domain of the 4-cysteine type. The human cDNA directs production in Escherichia coli of a 70-kDa protein that reacts with a monoclonal antibody directed against the 70-kDa subunit of human RP-A. The recombinant 70-kDa subunit, purified from bacteria, exhibits single-stranded DNA binding activity comparable to that of the complete RP-A complex. The 70-kDa subunit is able to substitute for the complete human RP-A complex in stimulating the activity of DNA polymerase alpha-primase on a poly(dA).oligo(dT) template. However, the 70-kDa subunit alone cannot substitute for the complete RP-A complex in SV40 DNA replication in vitro, suggesting an important functional role for the other subunits.

Amino Acid Sequence↗
PubMed1979

Cloning of the replication gene P of bacteriophage lambda: effects of increased P-protein synthesis on cellular and phage DNA replication.

A restriction fragment of lambdaDNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/lambdaE complement lambdaPam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/lambdaE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of lambdaDNA proceeds normally under these conditions. Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type lambda (lambdawt), acquire the ability to replicate lambdaPam80 phage but not lambdawt when they are transformed with a plasmid carrying the lambdaP gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of lambdawt phage when infected at a high multiplicity. lambdaPam80 phage does not multiply in these cells.

Coliphages↗
PubMed1978

Regional replication of the bacterial chromosome induced by derepression of prophage lambda. III. Role of the replication in escape synthesis of gal operon.

Recent evidence suggests that the escape synthesis of gal operon following derepression of the prophage lambda in Escherichia coli K12 involves transcription originating at the lambda promoter (PL) to extend through gal under the conditions in which lambda DNA replication is prevented. Whether the observed expression of gal is due to transcription initiating at PL or at the bacterial promoter for gal (Pgal) was examined in the case of lambda DNA replication being normal. The experiments are based on that two types of transcription are distinguished from each other by the following properties: 1. Pgal-promoted transcription is inhibited by chloramphenicol, while PL-promoted transcription is not. 2. PL-promoted transcription suppresses the polar effect caused by nonsense mutation in a bacterial gene, while Pgal-promoted transcription does not do so. -he results have suggested that gal escape synthesis in lambda-induced lysogen results from transcription which initiates not only at PL but also at Pgal. The Pgal-promoted transcription may be a consequence, direct or indirect, of the concomitant replication of gal DNA.

Chloramphenicol↗
PubMed1992

Transcription in vivo within the replication origin of the Escherichia coli chromosome: a mechanism for activating initiation of replication.

Within the replication origin, oriC, of the Escherichia coli chromosome, novel in vivo transcripts were detected which proceeded rightward and whose production was activated by DnaA protein. In contrast, DnaA protein repressed the previously described ori-L leftward transcription. The former should introduce negative supercoiling, and the latter positive supercoiling, into the 13-mers. The effects of transcription on the initiation of replication were also investigated by making constructs with promoters placed near oriC. Transcription was found to enhance the origin activity only when it was oriented in such a way as to introduce negative supercoiling into the 13-mers. From these results, we propose that transcription within oriC regulates replication initiation by altering the topology of the 13-mer region.

Base Sequence↗
PubMed1978

Analysis of DNA replication patterns of human fibroblast chromosomes: the replication map.

A replication map of human fibroblast chromosomes from two diploid human female fibroblast lines, 46,XX and 46,X, del (X)(q13), was determined using the 'fluorescent plus Giemsa' (FPG) technique. Each chromosome was found to stain homogeneously dark when thymidine was incorporated for the entire S phase of that particular cell. As the duration of exposure to thymidine progressively decreased by increasing the incubation time in bromodeoxyuridine, the staining intensity of chromosomes decreased and, concurrently, gaps in the staining began to appear. These gaps coincide with R bands and represent the earliest areas to complete DNA synthesis. As these areas widen and increase in frequency, first Q and G bands appear, and finally C bands. Homologous X chromosomes were easily differentiated by either a comparison of the bands present or their staining intensity. The replication kinetics of the structurally abnormal heterocyclic X chromosome were very similar to those of the normal heterocyclic X chromosome. The X chromosome with deletion of a portion of the long arm was consistently late in replication.

Cell Line↗
PubMed1976

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes. II. Replication of centromeric regions of chromosomes at the termination of the S period.

Replication of regions of chromosomes 1, 2, 3, 16, and group 4-5 was studied at the termination of the S period in primary cultures of embryonic fibroblasts (two embryos) and in cultures of peripheral blood (two women). Distinct differences were established in the pattern of late replication of the studied chromosomes in the cultures of the two types of cells. These differences consern first of all the centromeric and neighbouring regions of the chromosome. The content of late label in this region is 1.5-3 times higher in the cultures of fibroblasts than in the corresponding regions of leucocyte cultures. The difference is most pronounced in chromosomes 1, 3 and 16. It is suggested that the difference between cultures of these two types of cells in chromosome replication may be connected with the different genetic functioning of the centromeric and neighbouring regions in them. It is also possible that this difference is due to underreplication (or partial loss in an other way) of heterochromatin DNA of centromeric and neighbouring regions in leucocytes functioning for a long period without division.

Adult↗
PubMed1975

Initiation of DNA replication in Escherichia coli. III. Genetic analysis of the dna mutant exhibiting rifampicin-sensitive resumption of replication.

Temperature-sensitive mutants defective in the initiation of DNA replication are exposed to a non-permissive temperature to complete already initiated replication, and are transferred back to a permissive temperature. DNA synthesis can resume in the presence of rifampicin or rifampicin plus chloramphenicol in strain PC2 (dnaC2), but not in strain N167 (dna-167). In the presence of chloramphenicol alone, however, DNA synthesis can resume in both strains (Hirage and Saito, 1973, 1974). The double mutants carrying the dna-167 and dnaC2 mutations show the rifampicin-sensitive resumption of DNA replication as the dna-167 mutant. The rifampicin-sensitive character (designated as Rrr-) is closely linked with the temperature sensitivity of the dna-167 mutant in P1 transduction. The gene order is dna-167-tna-phoS-uncA-ilv. The Rrr- character does not correlate with the inactivation of the altered product of the mutated dna-167 gene at various temperatures in the double mutant carrying dna-167 and dnaC2. Although dnaC2 strains show the Rrr+ phenotype, the dnaC2 strains received the ilv-dnaA region of the Ts+ revertants obtained from a dna-167 strain show the Rrr- phenotype. These results suggest that the dna-167 mutant has two mutations which are closely linked to each other, controlling the Rrr- phenotype and the temperature sensitivity, respectively.

Bacterial Proteins↗
PubMed2025

Rad53 regulates RNase H1, which promotes DNA replication through sites of transcription-replication conflict.

RNA-DNA hybrids and R-loops can lead to extensive DNA damage and loss of genomic integrity if not regulated in a timely manner. Although RNase H1 overexpression is frequently used as a tool to resolve R-loops, the regulation of RNase H1, overexpressed or endogenous, remains poorly characterized. We reveal that in yeast, overexpressed RNase H1 (RNH1) has no effect on gene expression, cell growth, or RNA-DNA hybrid resolution in wild-type cells. Overexpressed RNase H1 does, however, remove RNA-DNA hybrids in mutants where hybrids have become dysregulated. Endogenous RNase H1 becomes up-regulated and chromatin-associated in the absence of Sen1 in a DNA replication checkpoint-dependent manner. Rnh1 gets recruited to genomic loci where RNA-DNA hybrids accumulate following the loss of Sen1. Rnh1, together with Sen1, promotes DNA replication at sites of transcription-replication conflict. Hence, RNase H1, overexpressed or endogenous, responds to unscheduled, stress-inducing RNA-DNA hybrids.

Ribonuclease H↗