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PubMed1989

Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages.

Cell lineages that give rise to the PNS were studied using the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) to visualized DNA replication immunocytochemically. The precursors of the PNS in the body segments of Drosophila embryos replicate their DNA in a spatially and temporally stereotyped pattern. The sequence of DNA replication within developing sensory organs suggests particular lineage relationships of the cells that constitute a sensory organ, i.e., neuron and associated support cells. In embryos that are mutant for the achaete-scute complex or daughterless, in which most or all of the PNS is missing, no BrdU-labeled cells were found in the appropriate regions, suggesting that these PNS precursors either do not form or fail to replicate. Thus, the BrdU technique allows determination as to whether a mutation affects the PNS precursors or terminal differentiation.

Animals↗

PubMed1995

Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication.

Initiation of in vitro ColE2 DNA replication requires the plasmid-specified Rep protein and DNA polymerase I but not RNA polymerase and DnaG primase. The ColE2 Rep protein binds specifically to the origin where replication initiates. Leading-strand synthesis initiates at a unique site in the origin and lagging-strand DNA synthesis terminates at another unique site in the origin. Here we show that the primer RNA for leading-strand synthesis at the origin has a unique structure of 5'-ppApGpA. We reconstituted the initiation reaction of leading-strand DNA synthesis by using purified proteins, the ColE2 Rep protein, Escherichia coli DNA polymerase I and SSB, and we showed that the ColE2 Rep protein is a priming enzyme, primase, which is specific for the ColE2 origin. The ColE2 Rep protein is unique among other primases in that it recognizes the origin region and synthesizes the primer RNA at a fixed site in the origin region. Specific requirement for ADP as a substrate and its direct incorporation into the 5' end of the primer RNA are also unique properties of the ColE2 Rep protein.

Adenosine Diphosphate↗

PubMed1981

Nucleotide requirements for DNA replication of SV40 chromatin in digitonin-treated and saponin-treated permeable cells.

Permeable cell systems have been developed by treatment with digitonin or saponin for studying in vitro DNA replication of chromatin. DNA replication of SV40 nucleoprotein complexes (SV40 chromatin) in these systems requires the addition of ATP, dCTP, dGTP, dTTP, and Mg2+. Further addition of three other ribonucleoside triphosphates slightly increased DNA synthesis. Omission of ATP greatly reduced DNA synthesis. In the presence of ATP, however, omission of dATP did not significantly alter or rather slightly enhanced DNA synthesis. Analysis of replicated SV40 DNA by gel electrophoresis and autoradiography indicated that complete replication of SV40 DNA and chromatin occurred in these systems

Animals↗

PubMed2003

DNA replication in the hyperthermophilic archaeon Sulfolobus solfataricus.

Studies of the DNA-replication machinery of Archaea have revealed striking similarities to that of eukaryotes. Indeed, it appears that in most cases Archaea possess a simplified version of the eukaryotic replication apparatus. Studies of Archaea are therefore shedding light on the fundamental processes of DNA replication in both domains of life.

Chromosomes, Archaeal↗

PubMed1996

Molecular design of inhibitors of in vitro oriC DNA replication based on the potential to block the ATP binding of DnaA protein.

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by binding to ATP. We earlier reported that 3-acetoxy-2,2'-bi-1H-indol inhibited the ATP binding to DnaA protein (Sasaki, S., Mizushima, T., Hashimoto, T., Maeda, M., and Sekimizu, K. (1994) Bioorg. Med. Chem. Lett. 4, 1771-1774). In the present study, derivatives of 3-acetoxy-2,2'-bi-1H-indol with different lengths of aliphatic chains at the 3-O position were synthesized, and their potential to inhibit the ATP binding to DnaA protein was examined. Elongation of the aliphatic chain resulted in inhibition of the ATP binding to DnaA protein at lower concentrations. Among the derivatives, 3-[N-(11-carboxyundecyl)]carbamoylmethoxy-2,2'-bi-1H-indol (structure 7 (3-CUCM-BI)) exhibited the most potent inhibition with an IC50 value of 7 microM. The mode of the inhibition was competitive. We further demonstrated that structure 7 (3-CUCM-BI) inhibited DNA replication of the oriC plasmid in a system reconstituted from purified proteins. This inhibition was specific for the initiation of DNA replication rather than for the elongation. The inhibition was overcome by preincubation of DnaA protein with ATP. Furthermore, structure 7 (3-CUCM-BI) showed little inhibition on DNA synthesis in the ABC primosome system. We propose that structure 7 (3-CUCM-BI) functions in the in vitro oriC DNA replication by inhibiting the ATP binding to DnaA protein.

Adenosine Triphosphate↗

PubMed2003

The papillomavirus E8-E2C protein represses DNA replication from extrachromosomal origins.

Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8-E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8-E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8-E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8-E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8-E2C (1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8-E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.

Base Sequence↗

PubMed1997

Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize.

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non-autonomous Ac derivative, Dissociation (Ds), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep+) and nonreplicating (rep-) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep+) or mutant, inactive derivatives of these genes (rep-). Excision of Ds was detected only in those cell nuclei co-transfected with the replicating Ds-vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 x 10(5)-fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.

Cell Nucleus↗

PubMed1994

A unique subpopulation of murine DNA polymerase alpha/primase specifically interacts with polyomavirus T antigen and stimulates DNA replication.

Murine cells or cell extracts support the replication of plasmids containing the replication origin (ori-DNA) of polyomavirus (Py) but not that of simian virus 40 (SV40), whereas human cells or cell extracts support the replication of SV40 ori-DNA but not that of Py ori-DNA. It was shown previously that fractions containing DNA polymerase alpha/primase from permissive cells allow viral ori-DNA replication to proceed in extracts of nonpermissive cells. To extend these observations, the binding of Py T antigen to both the permissive and nonpermissive DNA polymerase alpha/primase was examined. Py T antigen was retained by a murine DNA polymerase alpha/primase but not by a human DNA polymerase alpha/primase affinity column. Likewise, a Py T antigen affinity column retained DNA polymerase alpha/primase activity from murine cells but not from human cells. The murine fraction which bound to the Py T antigen column was able to stimulate Py ori-DNA replication in the nonpermissive extract. However, the DNA polymerase alpha/primase activity in this murine fraction constituted only a relatively small proportion (approximately 20 to 40%) of the total murine DNA polymerase alpha/primase that had been applied to the column. The DNA polymerase alpha/primase purified from the nonbound murine fraction, although far more replete in this activity, was incapable of supporting Py DNA replication. The two forms of murine DNA polymerase alpha/primase also differed in their interactions with Py T antigen. Our data thus demonstrate that there are two distinct populations of DNA polymerase alpha/primase in murine cells and that species-specific interactions between T antigen and DNA polymerases can be identified. They may also provide the basis for initiating a novel means of characterizing unique subpopulations of DNA polymerase alpha/primase.

Animals↗

PubMed2001

The Drosophila Geminin homolog: roles for Geminin in limiting DNA replication, in anaphase and in neurogenesis.

We have identified a Drosophila homolog of the DNA replication initiation inhibitor Geminin (Dm geminin) and show that it has all of the properties of Xenopus and human Geminin. During Drosophila development, Dm Geminin is present in cycling cells; protein accumulates during S phase and is degraded at the metaphase to anaphase transition. Overexpression of Dm geminin in embryos inhibits DNA replication, but cells enter mitosis arresting in metaphase, as in dup (cdt1) mutants, and undergo apoptosis. Overexpression of Dm Geminin also induces ectopic neural differentiation. Dm geminin mutant embryos exhibit anaphase defects at cycle 16 and increased numbers of S phase cells later in embryogenesis. In a partially female-sterile Dm geminin mutant, excessive DNA amplification in the ovarian follicle cells is observed. Our data suggest roles for Dm Geminin in limiting DNA replication, in anaphase and in neural differentiation.

Amino Acid Sequence↗

PubMed1987

Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.

Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA.

DNA Replication↗

PubMed1975

Meiosis in Coprinus VII. The prekaryogamy S-phase and the postkaryogamy DNA replication in C. lagopus.

The kinetics of incorporation of 32P into DNA have unequivocally shown that the premeiotic S-phase in Coprinus lagopus occurs before the onset of karyogamy. It takes 8 h under the control conditions (25 degrees C with a 16 h light-8 h dark regime) but only 6 h under the arrest-release conditions. An important discovery in this study is that the initiation of premeiotic DNA replication is subject to an arrest by restrictive conditions (35 degrees C under a continuous light regime) whereas that of the mitotic replication is not. Once initiated, meiotic DNA replication can continue even under the restrictive conditions. Incorporation of 32P into DNA at pachytene is quite extensive. These replications are considered to be repair replications.

Agaricales↗

PubMed1990

An essential arginine residue for initiation of protein-primed DNA replication.

A group of proteins that act as primers for initiation of linear DNA replication are called DNA-terminal proteins (terminal proteins). We have found a short stretch of conserved amino acid sequence among the terminal proteins from six different sources. The location of this sequence motif is also similar among the different terminal-proteins. To determine the functional role of this terminal-protein domain in DNA replication, we have studied the bacteriophage PRD1 system. The PRD1 terminal protein and DNA polymerase genes were cloned into expression vectors, and the recombinant plasmids were used for constructing PRD1 terminal protein mutants. Site-directed mutagenesis and functional analysis showed that one of the two arginines (Arg-174) in the conserved sequence is critical for the initiation complex-forming activity of the PRD1 terminal protein. Replacement of Arg-174 by noncharged amino acids resulted in nonfunctional terminal protein. Phenylglyoxal, an alpha-dicarbonyl compound that reacts with the guanidino group of arginine, inhibits initiation complex formation between PRD1 terminal protein and dGMP. On the basis of these results, we propose that Arg-174 represents, at least in part, the binding site for phosphate groups of dGTP.

Amino Acid Sequence↗

PubMed2003

The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeast dna2 mutants.

Bloom syndrome is a disorder of profound and early cancer predisposition in which cells become hypermutable, exhibit high frequency of sister chromatid exchanges, and show increased micronuclei. BLM, the gene mutated in Bloom syndrome, has been cloned previously, and the BLM protein is a member of the RecQ family of DNA helicases. Many lines of evidence suggest that BLM is involved either directly in DNA replication or in surveillance during DNA replication, but its specific roles remain unknown. Here we show that hBLM can suppress both the temperature-sensitive growth defect and the DNA damage sensitivity of the yeast DNA replication mutant dna2-1. The dna2-1 mutant is defective in a helicase-nuclease that is required either to coordinate with the crucial Saccharomyces cerevisiae (sc) FEN1 nuclease in Okazaki fragment maturation or to compensate for scFEN1 when its activity is impaired. We show that human BLM interacts with both scDna2 and scFEN1 by using coimmunoprecipitation from yeast extracts, suggesting that human BLM participates in the same steps of DNA replication or repair as scFEN1 and scDna2.

Adenosine Triphosphatases↗

PubMed1994

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

Animals↗

PubMed2000

Construction of a cosmid library of DNA replicated early in the S phase of normal human fibroblasts.

We constructed a subgenomic cosmid library of DNA replicated early in the S phase of normal human diploid fibroblasts. Cells were synchronized by release from confluence arrest and incubation in the presence of aphidicolin. Bromodeoxyuridine (BrdUrd) was added to aphidicolin-containing medium to label DNA replicated as cells entered S phase. Nuclear DNA was partially digested with Sau 3AI, and hybrid density DNA was separated in CsCl gradients. The purified early-replicating DNA was cloned into sCos1 cosmid vector. Clones were transferred individually into the wells of 96 microtiter plates (9,216 potential clones). Vigorous bacterial growth was detected in 8,742 of those wells. High-density colony hybridization filters (1, 536 clones/filter) were prepared from a set of replicas of the original plates. Bacteria remaining in the wells of replica plates were combined, mixed with freezing medium, and stored at -80 degrees C. These pooled stocks were analyzed by polymerase chain reaction to determine the presence of specific sequences in the library. Hybridization of high-density filters was used to identify the clones of interest, which were retrieved from the frozen cultures in the 96-well plates. In testing the library for the presence of 14 known early-replicating genes, we found sequences at or near 5 of them: APRT, beta-actin, beta-tubulin, c-myc, and HPRT. This library is a valuable resource for the isolation and analysis of certain DNA sequences replicated at the beginning of S phase, including potential origins of bidirectional replication.

Aphidicolin↗

PubMed1996

Two regions within the DNA binding domain of nuclear factor I interact with DNA and stimulate adenovirus DNA replication independently.

The cellular transcription factor nuclear factor I (NFI) stimulates adenovirus DNA replication by up to 50-fold. The NFI DNA binding domain (NFI-BD) is sufficient for stimulation and interacts with the viral DNA polymerase, thereby recruiting the precursor terminal protein-DNA polymerase complex (pTP-pol) to the origin of replication. The mechanism of DNA binding by NFI is unknown. To examine DNA binding and stimulation of adenovirus DNA replication by NFI-BD in more detail, we generated a series of deletion mutants and show that the DNA binding domain of NFI consists of two subdomains: a highly basic N-terminal domain that binds nonspecifically to DNA and a C-terminal domain that binds specifically but with very low affinity to the NFI recognition site. Both of these subdomains stimulate DNA replication, although not to the same extent as the intact DNA binding domain. The N-terminal domain has an alpha-helical structure, as shown by circular dichroism spectroscopy. The C-terminal domain interacts with the pTP-pol complex and is able to recruit the pTP-pol complex to DNA, which leads to pTP-pol-dependent stimulation of replication. The N-terminal domain also stimulates replication in a pTP-pol-dependent manner and enhances binding of pTP-pol to DNA. Since we could not detect a direct protein-protein interaction between pTP-pol and the N-terminal domain, we suggest that this domain stimulates replication by inducing structural changes in the DNA.

Adenoviruses, Human↗

PubMed2006

Kaposi's sarcoma-associated herpesvirus ori-Lyt-dependent DNA replication: dual role of replication and transcription activator.

Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral propagation and pathogenicity. In Kaposi's sarcoma lesions, constant lytic replication plays a role in sustaining the population of latently infected cells that otherwise are quickly lost by segregation of latent viral episomes as spindle cells divide. Lytic DNA replication initiates from an origin (ori-Lyt) and requires trans-acting elements. Two functional ori-Lyts have been identified in the KSHV genome. Some cis-acting and trans-acting elements for ori-Lyt-dependent DNA replication have been found. Among these, K8 binding sites, a cluster of C/EBP binding motifs, and a replication and transcription activator (RTA) responsive element (RRE) are crucial cis-acting elements. Binding of K8 and RTA proteins to these motifs in ori-Lyt DNA was demonstrated to be absolutely essential for DNA replication. In the present study, functional roles of RTA in ori-Lyt-dependent DNA replication have been investigated. Two distinct functions of RTA were revealed. First, RTA activates an ori-Lyt promoter and initiates transcription across GC-rich tandem repeats. This RTA-mediated transcription is indispensable for DNA replication. Second, RTA is a component of the replication compartment, where RTA interacts with prereplication complexes composed of at least six core machinery proteins and K8. The prereplication complexes are recruited to ori-Lyt DNA through RTA, which interacts with the RRE, as well as K8, which binds to a cluster of C/EBP binding motifs with the aid of C/EBP alpha. The revelation of these two functions of RTA, together with its role in initiation of a transcriptional cascade that leads to transcription of all viral lytic genes, shows that RTA is a critical initiator and regulator of KSHV lytic DNA replication and viral propagation.

Amino Acid Sequence↗

PubMed1994

The 3'-->5' exonucleases of both DNA polymerases delta and epsilon participate in correcting errors of DNA replication in Saccharomyces cerevisiae.

DNA polymerases II (epsilon) and III (delta) are the only nuclear DNA polymerases known to possess an intrinsic 3'-->5' exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase delta and epsilon 3'-->5' exonuclease-deficient mutants, pol3-01 and pol2-4, respectively. pol3-01 and pol2-4 increased spontaneous mutation rates by factors of the order of 10(2) and 10(1), respectively, measured as URA3 forward mutation and his7-2 reversion. Surprisingly, a double mutant pol2-4 pol3-01 haploid was inviable. This was probably due to accumulation of unedited errors, since a pol2-4/pol2-4 pol3-01/pol3-01 diploid was viable, with the spontaneous his7-2 reversion rate increased by about 2 x 10(3)-fold. Analysis of mutation rates of double mutants indicated that the 3'-->5' exonucleases of DNA polymerases delta and epsilon can act competitively and that, like the 3'-->5' exonuclease of DNA polymerase delta, the 3'-->5' exonuclease of DNA polymerase epsilon acts in series with the PMS1 mismatch correction system. Mutational spectra at a URA3 gene placed in both orientations near to a defined replication origin provided evidence that the 3'-->5' exonucleases of DNA polymerases delta and epsilon act on opposite DNA strands, but were in sufficient to distinguish conclusively between different models of DNA replication.

Amino Acid Sequence↗