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At least 361 records · Page 20Linked to original sources
PubMed1984

Identification of the replicative intermediates in SV40 DNA replication in vitro.

The soluble replication system is which the exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro, has been developed (Ariga and Sugano, J.Virol. 48, 481, 1983). This paper further characterized the in vitro products synthesized on the cloned DNA containing the origin of SV40 DNA replication. The time course and pluse-chase experiments showed that the in vitro products were converted from the open circle to closed circles having the various superhelical densities, and finally to the twisted formI DNA seen in vivo by the analysis of agarose gel electrophoresis, alkaline sucrose gradient centrifugation, and density-transfer in isopycnic centrifugation. The replicative intermediates isolated after the short term incubation had replicated strands of the size smaller than the full length, most of which correspond to that of the putative Okazaki fragment. These and the previous results indicate that this in vitro system should be useful to investigate the molecular mechanism of SV40 DNA replication.

DNA Replication↗
PubMed2024

N6-methyladenosine modification of a parvovirus-encoded small noncoding RNA facilitates viral DNA replication through recruiting Y-family DNA polymerases.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) η and Pol κ likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.

Humans↗
PubMed1989

Repression of polyoma virus DNA replication by 5'-flanking region of mouse DNA polymerase beta gene containing transcriptional silencer elements.

Dual cis-acting silencer elements are located upstream of the mouse DNA polymerase beta gene (Yamaguchi, M., Hayashi, Y., and Matsukage, A. (1989) J. Biochem. (Tokyo) 105, 79-83). In order to examine possible involvement of transcriptional silencer elements in the regulation of DNA replication, we have utilized a transient replication system of the plasmid DNA carrying replication origin of polyoma virus DNA in mouse MOP8 cells, which is constitutively producing polyoma virus large T-antigen. The polyoma virus origin of DNA replication is composed of three cis-acting genetic elements called alpha, beta, and core, in which alpha and beta elements correspond to enhancer domains. When the 5'-flanking regions of the DNA polymerase beta gene containing silencer elements were placed at the late gene border of alpha element, they effectively repressed the DNA replication. However, when placed at the early gene border of core element, it only marginally repressed the DNA replication. These results suggest that the silencer elements at cis position repress polyoma virus DNA replication by impeding the enhancer function that activates the DNA replication.

Animals↗
PubMed1986

Deoxyribonuclease I sensitivity of DNA replicated in permeable mouse sarcoma cells.

To study chromatin structure at the sites of DNA replicated in permeable cells, deoxyribonuclease I (DNase I) sensitivity of newly replicated DNA in permeable mouse sarcoma cells was compared with that of newly replicated DNA in intact cells. About 35% of the DNA replicated in permeable cells was hypersensitive to DNase I, and the remaining DNA showed the same DNase I sensitivity as that of parental chromatin DNA. The sensitivity of DNA replicated in permeable cells was higher than that of DNA newly replicated in intact cells, and was close to that of DNA replicated in the presence of cycloheximide. The sensitivity of DNA pulse-labeled with [3H]deoxythymidine triphosphate by replication in permeable cells was reduced significantly by chasing with cold deoxythymidine triphosphate. The present results suggest that chromatin structure at the sites of DNA replicated in permeable cells is similar to that at the sites of DNA replicated in living cells in the absence of protein synthesis, and that some structural change (possibly toward the maturation) of newly replicated chromatin occurs after the DNA replication in permeable cells.

Animals↗
PubMed2004

Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts.

In eukaryotes, prereplication complexes (pre-RCs) containing ORC, Cdc6, Cdt1, and MCM2-7 are assembled on chromatin in the G1 phase. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period. As a result, DNA replication is limited to a single round per cell cycle. One inhibitor of pre-RC assembly, geminin, was discovered in Xenopus, and it binds and inactivates Cdt1 in S phase. However, removal of geminin from Xenopus egg extracts is insufficient to cause rereplication, suggesting that other safeguards against rereplication exist. Here, we show that Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the course of the first round of DNA replication in Xenopus egg extracts. Degradation depends on Cdk2/Cyclin E, Cdc45, RPA, and polymerase alpha, demonstrating a requirement for replication initiation. Cdt1 is ubiquitinated on chromatin, and this process also requires replication initiation. Once replication has initiated, Cdk2/Cyclin E is dispensable for Cdt1 degradation. When fresh Cdt1 is supplied after the first round of DNA replication, significant rereplication results, and rereplication is enhanced in the absence of geminin. Our results identify a replication-dependent proteolytic pathway that targets Cdt1 and that acts redundantly with geminin to inactivate Cdt1 in S phase.

Animals↗
PubMed1978

[DNA replication in HeLa cells after gamma irradiation. I. The period of DNA replicative synthesis after the irradiation of cells in the G1 phase with gamma rays in large doses].

HeLa G-63 cells irradiated by 5 krads of 60 Co-gamma-rays during their G1-period pass through the S-period of the same interphase just as do non-irradiated cells: the kinetics of labeled cells upon a 30 min incubation with 3H-TdR (radioautography) is the same for both irradiated and non-irradiated cell populations; the rate of 3H-thymidine incorporation during the S-period upon a 3H-TdR continuous incubation is the same for both the cell populations; the mean DNA content per nucleus of irradiated cell is doubled after the S-period (cytophotometry). With increasing the radiation dose (from 10 to 30 krads), the rate of 3H-TdR incorporation into cells falls, and the S-period is prolonged.

Cell Cycle↗
PubMed1989

Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro.

The purification and characterization of a replication-dependent chromatin assembly factor (CAF-I) from the nuclei of human cells is described. CAF-I is a multisubunit protein that, when added to a crude cytosol replication extract, promotes chromatin assembly on replicating SV40 DNA. Chromatin assembly by CAF-I requires and is coupled with DNA replication. The minichromosomes assembled de novo by CAF-I consist of correctly spaced nucleosomes containing the four core histones H2A, H2B, H3, and H4, which are supplied in a soluble form by the cytosol replication extract. Thus, by several criteria, the CAF-I-dependent chromatin assembly reaction described herein reflects the process of chromatin formation during DNA replication in vivo.

Biological Assay↗
PubMed2004

DNA replication in thermophiles.

DNA replication enzymes in the thermophilic Archaea have previously attracted attention due to their obvious use in methods such as PCR. The proofreading ability of the Pyrococcus furiosus DNA polymerase has resulted in a commercially successful product (Pfu polymerase). One of the many notable features of the Archaea is the fact that their DNA processing enzymes appear on the whole to be more like those found in eukaryotes than bacteria. These proteins also appear to be simpler versions of those found in eukaryotes. For these reasons, archaeal organisms make potentially interesting model systems to explore the molecular mechanisms of processes such as DNA replication, repair and recombination. Why archaeal DNA-manipulation systems were adopted over bacterial systems by eukaryotic cells remains a most interesting question that we suggest may be linked to thermophily.

Archaea↗
PubMed1997

The initiation of simian virus 40 DNA replication in vitro.

DNA replication is a complicated process that is largely regulated during stages of initiation. The Siman Virus 40 in vitro replication system has served as an excellent model for studies of the initiation of DNA replication, and its regulation, in eukaryotes. Initiation of SV40 replication requires a single viral protein termed T-antigen, all other proteins are supplied by the host. The recent determination of the solution structure of the T-antigen domain that recognizes the SV40 origin has provided significant insights into the initiation process. For example, it has afforded a clearer understanding of origin recognition, T-antigen oligomerization, and DNA unwinding. Furthermore, the Simian virus 40 in vitro replication system has been used to study nascent DNA formation in the vicinity of the viral origin of replication. Among the conclusions drawn from these experiments is that nascent DNA synthesis does not initiate in the core origin in vitro and that Okazaki fragment formation is complex. These and related studies demonstrate that significant progress has been made in understanding the initiation of DNA synthesis at the molecular level.

Animals↗
PubMed1976

Characterization of an adenovirus early protein required for viral DNA replication: a single strand specific DNA binding proteins.

1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.

Adenoviridae↗
PubMed1986

Xeroderma pigmentosum variant and normal fibroblasts show the same response to the inhibition of DNA replication by benzo[a]pyrene-diol-epoxide-I.

Xeroderma pigmentosum (XP) variant cells are characterized by an abnormal pattern of replication of DNA damaged by 254 nm radiation (u.v.). To see whether benzo[a]pyrene-diol-epoxide-I (BPDE-I) elicits the same response, we have compared the effects of u.v. and BPDE-I on DNA replication in XP variant and normal skin fibroblasts. Doses of u.v. that only affected replicon initiation in normal cells, inhibited DNA strand growth in the XP variant. These results were confirmed by measurements of the rate of growth of single-stranded nascent DNA in cells synchronized at the beginning of the S phase. Identical analyses using BPDE-I, however, indicated that the two cell types were equally sensitive to the inhibitions of both replicon initiation and DNA strand growth. These results indicate that the XP variant phenotype cannot be recognized in vitro by the pattern of response of fibroblasts to the inhibition of DNA replication by BPDE-I.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Temperature-sensitive mutants of adenovirus single-stranded DNA-binding protein. Inability to support DNA replication is associated with an altered DNA-binding activity of the protein.

The adenovirus single-stranded DNA-binding protein (DBP) is an essential factor in viral DNA replication. Three temperature-sensitive (ts) adenoviruses (Ad2+ND1ts23, Ad2ts111A, and Ad5ts125) are known to have single amino acid substitutions in their DBPs that result in defective DNA replication at the nonpermissive temperature. To elucidate the mechanism(s) involved in the ts phenotype, we purified the three mutant DBPs and studied their DNA-binding properties and their ability to support DNA replication in an in vitro system. The results confirm that the three ts DBPs were incapable of supporting DNA replication at the nonpermissive temperature (40 degrees C). The defect was found at both the initiation and elongation steps of DNA replication. The 2-fold stimulation of pTP.dCMP formation by the DBP was lost by prior heating of the ts DBPs. The pronounced effect of the DBP on the early elongation process was severely diminished, but not abolished, by prior heating to 40 degrees C. The functional change at 40 degrees C was irreversible, as the ts DBPs preincubated at 40 degrees C were no longer active when assayed at 30 degrees C. Upon heating to 40 degrees C, all three ts DBPs lost their ability to bind to oligonucleotides, although they still retained some binding activity for large single-stranded DNAs such as M13 DNA. Thus, the inability of these three ts DBPs to support DNA replication is attributable to their altered DNA-binding properties.

Adenoviridae↗
PubMed1998

The evolutionarily conserved zinc finger motif in the largest subunit of human replication protein A is required for DNA replication and mismatch repair but not for nucleotide excision repair.

The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved zinc finger motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this motif (RPAm) cannot support SV40 DNA replication. We have now investigated the role of this motif in several steps of DNA replication and in two DNA repair pathways. RPAm associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases alpha and delta, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leading strand and later Okazaki fragments is impaired. In contrast, RPAm can function well during the incision step of nucleotide excision repair and in a full repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1-278 or 222-411) were not functional in nucleotide excision repair. We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. Neither the deletion mutants nor RPAm can support this reaction. Therefore, the zinc finger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch repair but not for nucleotide excision repair.

Binding Sites↗
PubMed2003

The DnaAcos allele of Escherichia coli: hyperactive initiation is caused by substitution of A184V and Y271H, resulting in defective ATP binding and aberrant DNA replication control.

Chromosomal DNA replication is regulated at the level of commitment to this biochemical pathway. In Escherichia coli, DnaA protein appears to regulate this process. A mutant form, DnaAcos, carrying four amino acid substitutions, is apparently defective in responding to regulatory signals, because it induces hyperactive initiation from the bacterial replication origin (oriC). In this report, the phenotype of hyperactive initiation is shown to be the result of two specific amino acid substitutions. One (A184V) immediately adjacent to a Walker A box (P loop motif) causes a defect in ATP binding (Carr and Kaguni, 1996, Mol Microbiol 20: 1307-1318). The second amino acid substitution (Y271H) appears to stabilize the activity of the mutant protein carrying the A184V substitution. The mutant protein carrying both amino acid substitutions (A184V + Y271H) is defective in modulating the frequency of initiation from oriC, as demonstrated by marker frequency analysis of oriC and a locus near the replication terminus. These results indicate that a defect in ATP binding results in aberrant control of DNA replication.

Adenosine Triphosphate↗
PubMed1979

Inceptor and origin of DNA replication in lambdoid coliphages. I. The lambda DNA minimal replication system.

In a pBR313-lambda dv hybrid plasmid system, stepwise deletion and serial cloning procedures have led to a functional dissection of the DNA replication region of lambdoid bacteriophages lambda, 434 and 21. A simple system for initiation of DNA replication has been detected within lambdoid replicator DNAs, which is active in the absence of several normal replication elements, including the origin of replication (ori) and product of gene O. This "minimal" (or "mini") initiation system depends on the p0 or substitute leftward promoter in conjunction with the newly discovered "inceptor" (ice) element, which is located within the cII gene. Even the fragments containing ori are unable to initiate replication in these hybrid plasmids as long as fragments containing ice are missing. The base sequence of ice resembles transcriptional terminators and it appears to control both termination of primer RNA and inception of daughter strand DNA synthesis. Initiation in the p0-ice mini system of lambda or 21 phages requires the gene P product. Hwever, mini replication of 434 DNA hybrid plasmids required neither O nor P proteins, although there are only two single-base changes in the 434 inceptor sequence. The mini system is repressed by the elements of the maximal lambda replication system, as described in the accompanying publication.

Bacteriophage lambda↗
PubMed1998

Role of the adenovirus DNA-binding protein in in vitro adeno-associated virus DNA replication.

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences.

Adenoviridae↗
PubMed1995

The role of the 34-kDa subunit of human replication protein A in simian virus 40 DNA replication in vitro.

Human replication protein A (RPA) is a three subunit protein complex involved in DNA replication, repair, and recombination. We investigated the role of the 34-kDa subunit (p34) of RPA in DNA replication by generating a series of p34 mutants. While deletion of the N-terminal domain of p34 prevented its phosphorylation by both cyclin-dependent kinase (Cdk) and DNA-dependent kinase, a double point mutant that lacks the major phosphorylation sites for Cdk could be phosphorylated by DNA-dependent kinase. In simian virus 40 (SV40) DNA replication, RPA containing either of these mutants functioned as efficiently as wild-type RPA. However, mutant RPA containing C-terminally deleted p34 was only marginally active. This indicates that the C-terminal region, but not the phosphorylation domain of p34, is necessary for RPA function in DNA replication. Furthermore, RPA containing the C-terminally deleted p34 mutant could stimulate DNA polymerase alpha, and bind to single-stranded DNAs but was limited in its ability to unwind DNA or interact with SV40 large T antigen (T Ag). These results suggest that RPA p34 interacts with SV40 T Ag during the initiation of SV40 DNA replication and may be necessary for DNA unwinding.

Base Sequence↗
PubMed1984

The coupling of DNA repair-recombination functions with DNA replication in bacteriophage T4: a new DNA repair mutant.

The requirement of DNA repair-recombination functions for T4 phage DNA replication has been known for some time but the underlying basis for this relationship has been unclear. This report is concerned with a new uv-sensitive gene [uvsU], whose function appears to bridge these two major activities of DNA. The [uvsU] mutant fails to complement [uvsX] mutants but uvsU maps in a region distinct from uvsX. Furthermore, the uvsU mutation specifically suppressed the DNA replication defect but not the uv sensitivity of the uvsX mutation. The previously discovered uvsW gene, whose mutations suppress the DNA replication defects of gene 59, 46, and 47 mutations, seems to have an analogous role. As a possible explanation for these observations, it is suggested that the uvsW and uvsU gene products (gps) couple the DNA repair-recombination and replication functions by controlling the entry of DNA intermediates from the replication pool into the DNA repair-recombination pathway. Furthermore the suppression data are interpreted to suggest that the gps uvsW, 59, 46, and 47 function together. Similarly the gps uvsU and uvsX may form a functional unit.

Base Sequence↗