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Effect of mitomycin C and 60Co gamma-irradiation on the replication of SV40 in cell lines of varying permissivity for SV40 replication.

The effects of mitomycin C and 60Co gamma-irradiation, which induce production of SV40 from SV40-transformed hamster cells, on the replication of superinfecting SV40 or virus DNA in cells varying in permissivity for SV40 replication have been examined. These agents enhance replication of SV40 in an uninducible line of SV40-transformed hamster kidney cells and in nonpermissive secondary hamster kidney cells. The same treatments do not affect SV40 replication in semipermissive hamster (BHK21) and human (HEL, HEK) cells and inhibit SV40 replication in permissive monkey (TC-7) cells. We conclude that forms of induction treatment, such as mitomycin C or 60Co gamma-irradiation, modify the expression of host cell factors which determine the level of permissivity for SV40 infection.

Animals

Replication of bacteriophage phiK duplex replicative-form DNA in dnaB and dnaC mutants of Escherichia coli.

We have directly tested the effects of host cell DNA synthesis mutations on bacteriophage phiK replicative-form (RF) DNA replication in vivo. We observed that phiK RF DNA replication continued at normal rates in both dnaB and dnaC mutant hosts under conditions in which the activities of the dnaB and dnaC gene products were shown to be markedly reduced. This suggests that these two host proteins are not essential for normal phiK RF DNA replication. In control experiments we observed markedly reduced rates of phiK RF DNA replication in temperature-sensitive dnaG and dnaE host mutants, indicating that the products of these genes are essential. Thus, the mechanism of DNA chain initiation in vivo on the duplex RF DNA templates of isometric phages such as phiK apparently is different from that on the similar templates of isometric phages such as phiX174. The implications of this difference are discussed in the text.

Bacterial Proteins

Plasmid replication functions. II. Cloning analysis of the repA replication region of antibiotic resistance plasmid R6-5.

R6-5 is a low copy number, conjugative, FII incompatibility group plasmid that has a molecular length of 102 kb and that specifies resistance against several antibiotics (chloramphenicol, fusidic acid, kanamycin, streptomycin and sulphonamide) and mercury salts. By means of in vitro cloning procedures, mini plasmids have been generated that contain a DNA segment from the essential region of R6-5 that is only 2.6 kb in length. This DNA segment, which consists of two PstI fragments that are adjacent in the parent plasmid, carries all genes and sequences required for the regulated replication and incompatibility properties of R6-5, including its origin of replication, OriV, an essential function that has been designated RepA, and the copy control function, Cop. Three different polypeptides, having monomer molecular weights of 23,000, 10,000 and 9,500 daltons, are synthesized in detectable quantities by minicells carrying pBR322 hybrid plasmids that contain DNA segments from the R6-5 essential region. A spontaneous deletion derivative of a pBR322 hybrid plasmid that carries the R6-5 origin of replication was isolated. Heteroduplex analysis of this derivative plasmid indicates that the deleted DNA segment carries the R6-5 replication origin and that its termini consist of short inverted repeat sequences.

DNA Polymerase I

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

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

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

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

Discontinuous replication of replicative form DNA from bacteriophage phiX174.

Bacteriophage phiX174 DNA has been labeled with short pulses of [3H]thymidine during synthesis of replicative form molecules in infected Escherichia coli HF4704 cells. The replicating phiX174 DNA was isolated and analyzed by sedimentation in an alkaline sucrose gradient. During a brief pulse (5 sec at 30 degrees), the radioactivity incorporated into the complementary strand was found in chains much shorter than one genome length. Of the radioactivity incorporated into the viral strand, two-thirds was in the short pieces and the rest was in chains of one genome length or longer. RNA attachment to the 5' end of both strand components of the nascent short pieces was shown by the appearance of spleen exonuclease-digestable nascent molecules after alkali treatment. These observations suggest that the viral as well as the complementary strand is synthesized by the discontinuous mechanism with RNA primers during replication of duplex phiX174 DNA.

Centrifugation, Density Gradient

Replication timing networks reveal a link between transcription regulatory circuits and replication timing control.

DNA replication occurs in a defined temporal order known as the replication timing (RT) program and is regulated during development, coordinated with 3D genome organization and transcriptional activity. However, transcription and RT are not sufficiently coordinated to predict each other, suggesting an indirect relationship. Here, we exploit genome-wide RT profiles from 15 human cell types and intermediate differentiation stages derived from human embryonic stem cells to construct different types of RT regulatory networks. First, we constructed networks based on the coordinated RT changes during cell fate commitment to create highly complex RT networks composed of thousands of interactions that form specific functional subnetwork communities. We also constructed directional regulatory networks based on the order of RT changes within cell lineages, and identified master regulators of differentiation pathways. Finally, we explored relationships between RT networks and transcriptional regulatory networks (TRNs) by combining them into more complex circuitries of composite and bipartite networks. Results identified novel trans interactions linking transcription factors that are core to the regulatory circuitry of each cell type to RT changes occurring in those cell types. These core transcription factors were found to bind cooperatively to sites in the affected replication domains, providing provocative evidence that they constitute biologically significant directional interactions. Our findings suggest a regulatory link between the establishment of cell-type-specific TRNs and RT control during lineage specification.

Cell Differentiation

Replication of M-13 DNA in plasmolysed Escherichia coli cells. Structure of a replicative intermediate with restricted binding of intercalating dyes.

DNA molecules with restricted binding of intercalating dyes are observed as replicative intermediates during the replication of bacteriophage M-13 duplex DNA in a cellular system in vitro prepared by plasmolysis of M-13-am5-infected Escherichia coli cells. Restriction of dye binding is abolished by heating the DNA to 80 degrees C, but can be recovered by slow cooling of the heat-treated DNA. Radioactive pulse-label incorporated by these molecules is found exclusively in elongated viral strands of more than one genome length. In the electron microscope this DNA fraction is seen to contain a significant number of duplex DNA rings with two single-stranded tails protruding from the same region of the ring. It is proposed that these structures arise by branch migration during the isolation of replicating molecules containing only one single-stranded tail. The topological constraint in these molecules is most likely caused by base-pairing between partially complementary regions of the two single-stranded tails.

Binding Sites

Initiation and termination of deoxyribonucleic acid replication in bacteria after a stepwise increase in the velocity of replication.

The theoretical relations between replication, initiation, termination, and deoxyribonucleic acid (DNA) accumulation were derived for experiments in which the length of the time required for the replication of the bacterial chromosome (C period) can be varied. This theory enables one to determine absolute values of the C period from kinetics of DNA accumulation after a "stepup" with thymine-requiring bacteria that are subjected to a sudden increase in the exogenous thymine concentration. Application of this method of data evaluation to an observed step-up experiment with a thy-derivative of Escherichia coli B/r (ATCC 12407) indicated that the theory describes the observed post-step accumulation of DNA accurately within experimental errors. It is also concluded that changes in the replication velocity (C) do not measurably affect the timing of initiation events in a culture.

DNA Replication

Host cell DNA chain initiation protein requirements for replication of bacteriophage G4 replicative-form DNA.

Bacteriophages G4ev1 and G4bs1 are simple temperature-resistant derivatives of wild-type G4 as demonstrated by restriction endonuclease analyses. The rate of replication of the duplex replicative-form DNA of these phages was normal in dnaB and dnaC mutants of the host, whereas the rate was markedly reduced in a dnaG host mutant at the restrictive temperature. We conclude that G4 duplex DNA replication requires the host cell dnaG protein, but not the dnaB and dnaC proteins. The reasons for the differences between our conclusions and those based on previously published data are documented and discussed.

Bacterial Proteins

Replication of bacteriophage M13. XIV. Differential inhibition of the replication of M13 and M13 miniphage in a mutant of Escherichia coli defective in the 5' leads to 3' exonuclease associated with DNA polymerase I.

Previous studies have shown that M13 single-strand synthesis is inhibited at nonpermissive temperature in Escherichia coli polAexl, a temperature-sensitive mutant defective in the 5' leads to 3' exonuclease activity of polymerase I (T.-C. Chen and D. S. Ray, J. Mol. Biol. 106:589-604, 1976). Under these conditions the formation of covalently closed replicative form (RF) molecules is greatly reduced, and miniature forms of RF accumulate. We show here that the accumulation of mini-RFs is the consequence of a differential inhibition of the replication of unit-length phage and preexisting miniphage rather than a de novo production of miniphage. Mini-RFs do not accumulate even after as many as nine cycles of growth in the mutant host infected only with unit-length phage. Mixed infections of the mutant host with plaque-purified unit-length phage and a single cloned miniphage show that discontinuities in the mini-RFs are joined with higher efficiency than are those contained in unit-length RFs. After a shift to nonpermissive temperature during single-strand synthesis in cells infected with plaque-purified phage alone, M13 RFs are found largely as RFII molecules (RF form having one or more single-strand discontinuities) containing only a single discontinuity in the viral strand. The inability of the accumulated unit-length RFII molecules to actively replicate may reflect the presence of either a bound protein or RNA primer on the 5' terminus of the viral strand and provides further support for the existence of distinct initiation and termination events in the synthesis of the viral strand.

Coliphages

The role of pre-replication and post-replication processes in mutation induction in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine.

Studies were carried out on the repair and fixation of premutational damage induced in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The studies employed a temperature-sensitive DNA elongation mutant (dna9) and its combinations with mutants defective in pyrimidine dimer excision (uvr1, uvr2) and in recombination (rec1). The dna9 mutant is shown to be leaky, allowing about 1% of the normal rate of DNA synthesis at the restrictive temperature. Repair of premutational lesions was detected by a decline in mutation frequency with increasing delay in DNA replication in dna9 at the restrictive temperature. This repair is unaffected by the pyrimidine dimer excision system. Mutation fixation was detected by the ability of DNA from treated and then lysed cells to transfer mutants to recipient cells by transformation. Some fixation occurred at the restrictive temperature but much less than at the non-restrictive temperature suggesting that an appreciable minority of the mutations resulted from lesions introduced near the replication fork but that the majority of mutations arise from lesions introduced at some distance from the fork, perhaps randomly. The DNA synthesized immediately after MNNG treatment is of lower molecular weight than normal and returns to normal with time. This return is blocked in the rec1 mutant, suggesting that recombination is involved. The possible role of this process in MNNG mutagenesis is discussed.

DNA Repair

Virus-replicating T cells in the immune response of mice. III. Role of vesicular stomatitis virus-replicating T cells in the antibody response.

The functional role of the T cell (Tv) which can replicate vesicular stomatitis virus (VSV) on activation by the antigen was investigated in antibody response in vitro. By the inoculation of VSV into the culture, marked augmentation of antibody response to sheep erythrocytes (SRBC) was observed in the culture of spleen cells taken more than 3 days after the immunization with SRBC, suggesting that the VSV-susceptible suppressor cells were included in these spleen cells and the activity was eliminated by the effect of VSV. Development of two distinct types of suppressor T cells was revealed in the spleen of mice after the priming with SRBC. First, nylon wool nonadherent (NAd) suppressor T cells found in the spleen cells taken 3 days after immunization, and second, nylon wool adherent (Ad) suppressor T cells found in the spleen cells taken approximately 1 wk after immunization. The activity of nylon Ad suppressor T cells was completely abolished by VSV-preinfection, whereas that of nylon NAd suppressor T cells was unaffected. It was also shown that the helper T-cell activity was not influenced by VSV-preinfection. These results provided direct evidence that nylon Ad suppressor T cell but not nylon NAd suppressor T cell nor helper T cell can actually replicate VSV after antigenic stimulation. Thus it was strongly suggested that Tv represents the nylon Ad suppressor T cells.

Animals

Replication of polyoma DNA in isolated nuclei. V. Complementation of in vitro DNA replication.

Nuclei from polyoma-infected 3T6 fibroblasts elongate in vitro the progeny strands of the replicative intermediates of polyoma DNA. When high concentrations of such nuclei were incubated, short DNA fragments were formed and subsequently added onto growing progeny strands. When nuclei were repeatedly washed with buffer containing detergent and then incubated at low concentrations. DNA synthesis was decreased. In particular, the joining process was reduced, resulting in an accumulation of short DNA fragments. All aspects of the synthetic capacity of the nuclei were restored by addition of cytoplasmic extract. Additions of purified enzymes (polynucleotide ligase from calf thymus or Escherichia coli together with E. coli DNA polymerase I) increased the joining function of the nuclei. The system can be used for the identification of the enzymatic steps concerned with polyoma DNA replication.

Animals

In situ replication techniques: I. Preliminary screening and the negative replication technique.

Fourteen commercially available elastomeric materials were investigated for their suitability as in vivo replication media. After a preliminary screening on an annotated specimen, three materials were chosen for further study. Of all materials evaluated, Xantopren Blue and Silene silicone impression materials provided the best results in vivo. This study demonstrates that a high resolution (ca 1 micron) negative replication technique may prove useful for the clinical evaluation of single-surface phenomena.

Acetates

Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.

Adenosine Triphosphate