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At least 685 records · Page 38Linked to original sources
PubMed1983

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells.

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.

Animals↗
PubMed2005

The MCM complex: its role in DNA replication and implications for cancer therapy.

The MCM complex controls the once per cell cycle DNA replication in eukaryotic cells. In a process known as DNA replication licensing, it primes chromatin for DNA replication by binding origins of DNA replication during the late M to early G1 phase of the cell cycle. Activated by S phase promoting protein kinases, the origin-bound MCM complexes unwind the double stranded DNA at the origins, recruit DNA polymerases and initiate DNA synthesis. Coupled with the initiation of DNA replication in the S phase, the MCM complexes move away from replication origins as a component of the DNA replication fork, likely serving as DNA helicases. Their departure deprives replication origins the ability to re-initiate DNA replication for the reminder of the cell cycle. Because of its vital role in genome duplication in proliferating cells, deregulation of the MCM function results in chromosomal defects that may contribute to tumorigenesis. The MCM proteins are highly expressed in malignant human cancers cells and pre-cancerous cells undergoing malignant transformation. They are not expressed in differentiated somatic cells that have been withdrawn from the cell cycle. Therefore, these proteins are ideal diagnostic markers for cancer and promising targets for anti-cancer drug development. In this article, I will overview the structures and functions of the MCM complex with an effort to integrate insights from recent biochemical and structural studies. Discussions will also cover activities and structures of the complex that may be useful for the development of drug screens.

Animals↗
PubMed1983

The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12.

The sdrA224 mutants of Escherichia coli K-12, capable of continued DNA replication in the absence of protein synthesis (stable DNA replication), tolerate inactivation of the dnaA gene by insertion of transposon Tn10. Furthermore, oriC, the origin of E. coli chromosome replication, can be deleted from the chromosome of sdrA mutants without loss of viability. The results suggest the presence of a second, normally repressed, initiation system for chromosome replication alternative to the 'normal' dnaA+ oriC+-dependent initiation mechanism.

Bacterial Proteins↗
PubMed2006

Structural analysis of the human neuroblastoma DNA replication complex: insights into faulty proliferation.

BACKGROUND: Whether faulty DNA synthesis contributes to neuroblastoma (NB) pathogenesis has received little attention. We have shown that NB DNA replication is orchestrated by a multiprotein DNA replication complex (DNA synthesome) which mediates error-prone DNA synthesis. We sought to define proteomic alterations of the NB DNA synthesome, which lead to lowered replication fidelity. METHODS: DNA synthesomes from NB and neural stem (NS) cell culture were purified. Proteomic differences of the DNA synthesome between NB and NS cells were determined using 2-dimensional polyacrylamide gel electrophoresis and immunodetection. RESULTS: DNA synthesome proteins from NB and NS cells were compared. Only replication protein A (RPA) showed distinct changes. RPA from NS cells resolved as a single spot (isoelectric point [pI] 5.75), whereas RPA in NB showed a main spot (pI 5.75) along with 3 additional isoforms. RPA binds to single-stranded DNA and participates in protein-protein interactions. CONCLUSIONS: NB DNA synthesome showed 3 distinct isoforms of RPA when compared with NS cells. These findings are significant in that it is possible to link changes in the fidelity of DNA replication with a specific protein alteration of the NB DNA synthetic apparatus. The novel RPA forms may be a new signature of NB malignancy.

Blotting, Western↗
PubMed1980

Differential effects of aphidicolin on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells.

Aphidicolin clearly discriminated replicative DNA synthesis from unscheduled DNA synthesis. Aphidicolin inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells. The mode of inhibition of aphidicolin was a mixed type with respect to deoxycytidine triphosphate but was non-competitive with respect to the other three deoxynucleoside triphosphates. Aphidicolin did not affect the activity of unscheduled DNA synthesis in either bleomycin-treated permeable sarcoma cells or isolated rat liver nuclei. Considering the difference in sensitivity of DNA polymerase alpha and beta to aphidicolin, and other related information reported previously, the results are compatible with the idea that DNA polymerase alpha is involved in replicative DNA synthesis and DNA polymerase beta in unscheduled DNA synthesis in the present systems.

Animals↗
PubMed2000

Disruption of nuclear lamin organization blocks the elongation phase of DNA replication.

The role of nuclear lamins in DNA replication is unclear. To address this, nuclei were assembled in Xenopus extracts containing AraC, a reversible inhibitor that blocks near the onset of the elongation phase of replication. Dominant-negative lamin mutants lacking their NH(2)-terminal domains were added to assembled nuclei to disrupt lamin organization. This prevented the resumption of DNA replication after the release of the AraC block. This inhibition of replication was not due to gross disruption of nuclear envelope structure and function. The organization of initiation factors was not altered by lamin disruption, and nuclei resumed replication when transferred to extracts treated with CIP, an inhibitor of the cyclin-dependent kinase (cdk) 2-dependent step of initiation. This suggests that alteration of lamin organization does not affect the initiation phase of DNA replication. Instead, we find that disruption of lamin organization inhibited chain elongation in a dose-dependent fashion. Furthermore, the established organization of two elongation factors, proliferating cell nuclear antigen, and replication factor complex, was disrupted by DeltaNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.

Animals↗
PubMed2000

Dynamics of DNA replication factories in living cells.

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.

Animals↗
PubMed2006

OriDB: a DNA replication origin database.

Replication of eukaryotic chromosomes initiates at multiple sites called replication origins. Replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where several complementary studies have mapped their locations genome-wide. We have collated these datasets, taking account of the resolution of each study, to generate a single list of distinct origin sites. OriDB provides a web-based catalogue of these confirmed and predicted S.cerevisiae DNA replication origin sites. Each proposed or confirmed origin site appears as a record in OriDB, with each record comprising seven pages. These pages provide, in text and graphical formats, the following information: genomic location and chromosome context of the origin site; time of origin replication; DNA sequence of proposed or experimentally confirmed origin elements; free energy required to open the DNA duplex (stress-induced DNA duplex destabilization or SIDD); and phylogenetic conservation of sequence elements. In addition, OriDB encourages community submission of additional information for each origin site through a User Notes facility. Origin sites are linked to several external resources, including the Saccharomyces Genome Database (SGD) and relevant publications at PubMed. Finally, a Chromosome Viewer utility allows users to interactively generate graphical representations of DNA replication data genome-wide. OriDB is available at www.oridb.org.

Chromosomes, Fungal↗
PubMed2003

Characterization of novel reverse transcriptase and other RNA-associated catalytic activities by human DNA polymerase gamma: importance in mitochondrial DNA replication.

During mitochondrial DNA (mtDNA) replication, DNA/RNA heteroduplex intermediates are formed. To understand how and why ribonucleotides are involved in mtDNA replication, we have studied the novel RNA-associated activities of human mitochondrial DNA polymerase (Pol gamma), including reverse transcription, RNA-directed 3' --> 5' DNA excision, RNA-primed DNA synthesis, and ribonucleotide incorporation. Remarkably, Pol gamma catalyzes reverse transcription with a slightly higher efficiency than HIV-1 reverse transcriptase, suggesting that the activity may be physiologically significant, and furthermore, proofreading activity with an RNA template was also observed. RNA-primed DNA synthesis activity is required for initiation of mtDNA replication, and we have found that Pol gamma holoenzyme is capable of performing this reaction at a physiologically relevant rate and that the accessory subunit plays an essential role in the initiation steps. Single ribonucleotides have been found scattered in the mtDNA genome, although their role and significance are not yet defined. Our finding that Pol gamma also incorporates ribonucleotide triphosphates into a DNA primer offers a plausible enzymatic pathway for the origin of the RNA-containing mtDNA genome.

Binding Sites↗
PubMed1994

Replication factor A is required in vivo for DNA replication, repair, and recombination.

Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.

DNA Mutational Analysis↗
PubMed2001

Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis.

We have used high-density DNA microarrays to provide an analysis of gene regulation during the mammalian cell cycle and the role of E2F in this process. Cell cycle analysis was facilitated by a combined examination of gene control in serum-stimulated fibroblasts and cells synchronized at G(1)/S by hydroxyurea block that were then released to proceed through the cell cycle. The latter approach (G(1)/S synchronization) is critical for rigorously maintaining cell synchrony for unambiguous analysis of gene regulation in later stages of the cell cycle. Analysis of these samples identified seven distinct clusters of genes that exhibit unique patterns of expression. Genes tend to cluster within these groups based on common function and the time during the cell cycle that the activity is required. Placed in this context, the analysis of genes induced by E2F proteins identified genes or expressed sequence tags not previously described as regulated by E2F proteins; surprisingly, many of these encode proteins known to function during mitosis. A comparison of the E2F-induced genes with the patterns of cell growth-regulated gene expression revealed that virtually all of the E2F-induced genes are found in only two of the cell cycle clusters; one group was regulated at G(1)/S, and the second group, which included the mitotic activities, was regulated at G(2). The activation of the G(2) genes suggests a broader role for E2F in the control of both DNA replication and mitotic activities.

Animals↗
PubMed1994

Topoisomerase III, but not topoisomerase I, can support nascent chain elongation during theta-type DNA replication.

Topoisomerase III, but not topoisomerase I, could, in the absence of DNA gyrase, support bidirectional DNA replication in an oriC plasmid DNA replication system reconstituted with purified proteins. The initial rate of DNA synthesis and the efficiency of nascent chain elongation showed that topoisomerase III-stimulated DNA replication was as efficient as gyrase-stimulated DNA replication. In addition, topoisomerase III was also able, in the absence of DNA gyrase, to decatenate the replicating daughter DNA molecules to form monomer product. Thus, of the four topoisomerases in Escherichia coli, three, topoisomerases III and IV and DNA gyrase, can support nascent chain elongation, whereas only two, topoisomerases III and IV, can topologically resolve the daughter molecules.

DNA Replication↗
PubMed1995

Further characterization of the human cell multiprotein DNA replication complex.

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.

Blotting, Western↗
PubMed1988

Operon structure of dnaT and dnaC genes essential for normal and stable DNA replication of Escherichia coli chromosome.

dnaT and dnaC of Escherichia coli, whose products are components of the primosome, encode genes essential for both normal chromosomal replication and stable DNA replication induced by SOS signals. dnaT and dnaC together with an unknown gene (P-18), which encodes an 18-kDa polypeptide present downstream of dnaC, are cotranscribed from a promoter located 104 base pairs upstream of dnaT. A portion of the transcripts are processed 16 or 27 base pairs upstream of the dnaC coding region and 20% of the transcripts extending through dnaC are terminated at a terminator 103 base pairs downstream of dnaC. The rest of the transcripts pass through this terminator and are terminated downstream of the P-18 gene. The predicted amino acid sequence of the P-18 protein indicate the presence of a 26-amino acid signal peptide at its N terminus, suggesting that this protein may be secreted into the membrane or a periplasmic space. Another gene, which encodes a 14-kDa hydrophobic protein (P-14), was identified upstream of dnaT. The P-14 gene is transcribed from an upstream promoter, and its transcript extends through dnaT and dnaC. These results indicate that dnaT and dnaC constitute an operon with two unknown genes whose products may be associated with a membrane.

Amino Acid Sequence↗
PubMed1997

The DNA polymerase alpha-primase complex couples DNA replication, cell-cycle progression and DNA-damage response.

The highly conserved DNA polymerase alpha-primase complex (pol-prism) is the only eukaryotic DNA polymerase that can initiate DNA synthesis de novo. It is required both for the initiation of DNA replication at chromosomal origins and for the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. The dual role of pol-prim makes it a likely target for mechanisms that control cell-cycle S-phase entry and progression.

Animals↗
PubMed2003

Epigenomic replication: linking epigenetics to DNA replication.

The information contained within the linear sequence of bases (the genome) must be faithfully replicated in each cell cycle, with a balance of constancy and variation taking place over the course of evolution. Recently, it has become clear that additional information important for genetic regulation is contained within the chromatin proteins associated with DNA (the epigenome). Epigenetic information also must be faithfully duplicated in each cell cycle, with a balance of constancy and variation taking place during the course of development to achieve differentiation while maintaining identity within cell lineages. Both the genome and the epigenome are synthesized at the replication fork, so the events occurring during S-phase provide a critical window of opportunity for eliciting change or maintaining existing genetic states. Cells discriminate between different states of chromatin through the activities of proteins that selectively modify the structure of chromatin. Several recent studies report the localization of certain chromatin modifying proteins to replication forks at specific times during S-phase. Since transcriptionally active and inactive chromosome domains generally replicate at different times during S-phase, this spatiotemporal regulation of chromatin assembly proteins may be an integral part of epigenetic inheritance.

Animals↗
PubMed1993

Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen.

Initiation of cell-free simian virus 40 (SV40) DNA replication requires the interaction of DNA polymerase alpha/primase with a preinitiation complex containing the viral T antigen and cellular proteins, replication protein A, and topoisomerase I or II. To further understand the molecular mechanisms of the transition from preinitiation to initiation, the intermolecular interaction between human DNA polymerase alpha and T antigen was investigated. We have demonstrated that the human DNA polymerase alpha catalytic polypeptide is able to associate with SV40 large T antigen directly under physiological conditions. A physical association between these two proteins was detected by coimmunoprecipitation with monoclonal antibodies from insect cells coinfected with recombinant baculoviruses. A domain of human polymerase alpha physically interacting with T antigen was identified within the amino-terminal region from residues 195 to 313. This domain of human polymerase alpha was able to form a nonproductive complex with T antigen, causing inhibition of the SV40 DNA replication in vitro. Kinetics of the inhibition indicated that this polymerase domain can inhibit viral replication only during the preinitiation stage. Extra molecules of T antigen could partially overcome the inhibition only prior to initiation complex formation. The data support the conclusion that initiation of SV40 DNA replication requires the physical interaction of T antigen in the preinitiation complex with the amino-terminal domain of human polymerase alpha from amino acid residues 195 to 313.

Antigens, Viral, Tumor↗