Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA replication”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources
PubMed2003

Kaposi's sarcoma-associated herpesvirus lytic origin (ori-Lyt)-dependent DNA replication: identification of the ori-Lyt and association of K8 bZip protein with the origin.

Herpesviruses utilize different origins of replication during lytic versus latent infection. Latent DNA replication depends on host cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin binding protein (OBP) that recruits the core replication machinery. In this report, we demonstrated that DNA sequences in two noncoding regions of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome, between open reading frames (ORFs) K4.2 and K5 and between K12 and ORF71, are able to serve as origins for lytic cycle-specific DNA replication. The two ori-Lyt domains share an almost identical 1,153-bp sequence and a 600-bp downstream GC-rich repeat sequence, and the 1.7-kb DNA sequences are sufficient to act as a cis signal for replication. We also showed that an AT-palindromic sequence in the ori-Lyt domain is essential for the DNA replication. In addition, a virally encoded bZip protein, namely K8, was found to bind to a DNA sequence within the ori-Lyt by using a DNA binding site selection assay. The binding of K8 to this region was confirmed in cells by using a chromatin immunoprecipitation method. Further analysis revealed that K8 binds to an extended region, and the entire region is 100% conserved between two KSHV ori-Lyt's. K8 protein displays significant similarity to the Zta protein of Epstein-Barr virus (EBV), which is a known OBP of EBV. This notion, together with the ability of K8 to bind to the KSHV ori-Lyt, suggests that K8 may function as an OBP in KSHV.

Base Sequence↗
PubMed2005

Effects of RNA interference of Trypanosoma brucei structure-specific endonuclease-I on kinetoplast DNA replication.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid protozoa, is a network containing several thousand topologically interlocked DNA minicircles. Kinetoplast DNA synthesis involves release of minicircles from the network, replication of the free minicircles, and reattachment of the progeny back onto the network. One enzyme involved in this process is structure-specific endonuclease-I. This enzyme, originally purified from Crithidia fasciculata, has been proposed to remove minicircle replication primers (Engel, M. L., and Ray, D. S. (1998) Nucleic Acids Res. 26, 4773-4778). We have studied the structure-specific endonuclease-I homolog from Trypanosoma brucei, showing it to be localized in the antipodal sites flanking the kinetoplast DNA disk, as previously shown in C. fasciculata. RNA interference of structure-specific endonuclease-I caused persistence of a single ribonucleotide at the 5' end of both the leading strand and at least the first Okazaki fragment in network minicircles, demonstrating that this enzyme in fact functions in primer removal. Probably because of the persistence of primers, RNA interference also impeded the reattachment of newly replicated free minicircles to the network and caused a delay in kinetoplast DNA segregation. These effects ultimately led to shrinkage and loss of the kinetoplast DNA network and cessation of growth of the cell.

Animals↗
PubMed2006

Distinctive activities of DNA polymerases during human DNA replication.

The contributions of human DNA polymerases (pols) alpha, delta and epsilon during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol delta was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols alpha and epsilon remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol delta, unlike pols alpha and epsilon, increased in the late S phase. Moreover, pol delta neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol epsilon were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol epsilon to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol delta was mainly dispersed on fibrous structures. Pol alpha and proliferating cell nuclear antigen displayed partial colocalization with pol delta and epsilon, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols delta and epsilon pursue their functions at least partly independently during DNA replication.

Chromatin↗
PubMed1998

Transcription factor E2F and cyclin E-Cdk2 complex cooperate to induce chromosomal DNA replication in Xenopus oocytes.

Although no chromosomal DNA replication actually occurs during Xenopus oocyte maturation, the capability develops during the late meiosis I (MI) phase in response to progesterone. This ability, however, is suppressed by Mos proteins and maturation/mitosis promoting factor during the second meiosis phase (meiosis II; MII) until fertilization. Inhibition of RNA synthesis by actinomycin D during early MI prevented induction of the replication ability, but did not interfere with initiation of the meiotic cell cycle progression characterized by oscillation of the maturation/mitosis promoting factor activity and germinal vesicle breakdown. Microinjection of recombinant proteins such as dominant-negative E2F or universal Cdk inhibitors, p21 and p27, but not wild type human E2F-1 or Cdk4-specific inhibitor, p19, into maturing oocytes during MI abolished induction of the DNA replication ability. Co-injection of human E2F-1 and cyclin E proteins into immature oocytes allowed them to initiate DNA replication even in the absence of progesterone treatment. Injection of cyclin E alone, which was sufficient to activate endogenous Cdk2 kinase, failed to induce DNA replication. Moreover, the activation of Cdk2 was not affected under the conditions where DNA replication was blocked by actinomycin D. Thus, like somatic cells, both activities of E2F and cyclin E-Cdk2 complex are required for induction of the DNA replication ability in maturing Xenopus oocytes, and enhancement of both activities enables oocytes to override DNA-replication inhibitory mechanisms that specifically lie in maturing oocytes.

Animals↗
PubMed1996

The human cell multiprotein DNA replication complex (MRC): the effect of camptothecin on its ability to support in vitro DNA synthesis.

PURPOSE: We have previously reported on the isolation and characterization of a multiprotein DNA replication complex (MRC) from HeLa cells that fully supports in vitro DNA replication. Based upon its ability to replicate DNA in a cell-free environment (devoid of other cellular processes) the MRC may serve as a unique model system for investigating the mechanisms of action of anticancer drugs that directly affect DNA synthesis. The experiments described in this report were performed to establish whether the MRC could serve as a model system to examine in detail the mechanism of action of camptothecin, a DNA topoisomerase I inhibitor. METHODS: We examined the effects of increasing concentrations of camptothecin on HeLa cell survival, intact HeLa cell DNA synthesis and MRC-mediated in vitro DNA replication. We also performed topoisomerase I assays in the presence of increasing concentrations of camptothecin to study the direct effects of the agent on MRC-associated topoisomerase I activity. Furthermore, we employed an SDS precipitation assay to measure the formation of MRC-associated topoisomerase I-cleavable complexes in the presence of increasing concentrations of camptothecin. RESULTS: We found a close correlation between the IC50 values for intact HeLa cell DNA synthesis (0.15 microM) and MRC-mediated in vitro DNA synthesis (0.05 microM). Similarly, we found that 0.05 microM camptothecin inhibited MRC-associated topoisomerase I activity by approximately 50%. In addition, we found that the formation of MRC-associated topoisomerase I-cleavable complexes increased linearly with increasing concentrations of camptothecin. CONCLUSIONS: The data presented in this report support the use of the MRC as a model system to study the mechanism of action of camptothecin. We anticipate that future studies with the MRC will help elucidate the cellular consequences of camptothecin-cleavable complex formation.

Antineoplastic Agents, Phytogenic↗
PubMed1984

Regulation of a new bacteriophage T4 gene, 69, that spans an origin of DNA replication.

We have determined the DNA sequence and transcription patterns in a 3-kb segment (between 15 and 18 kb on the standard phage T4 map) spanning an origin of DNA replication. A new gene, 69, spans this origin. Gene 69 codes for two overlapping proteins that share a common C-terminal segment. Defective DNA replication in an appropriate amber mutant shows that at least the larger of the two proteins is required for efficient T4 DNA replication. The two proteins coded by gene 69 are expressed from different transcripts that are under different regulation. The smaller protein, gp69*, can be expressed immediately from an Escherichia coli-like promoter, whereas expression of the larger protein, gp69, must be delayed since its middle promoter requires T4 coded proteins, most likely gp mot, for activation. We discuss the possible significance of two overlapping proteins in the assembly of replisomes. Gene 69 is bracketed by the non-essential early gene dam (DNA adenine methylase) and the late gene soc (small outer capsid protein). Transcripts through this region are interdigitated in a complex pattern, which reveals all elements that are thought to be important in regulation of pre-replicative and post-replicative T4 genes.

Amino Acid Sequence↗
PubMed1996

Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast.

In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase. In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G1 is necessary for subsequent initiation of DNA replication triggered by activation in late G1 of Cdc28/Cdk1 kinases associated with B-type cyclins Clb1-Clb6. The absence of pre-RCs during G2 and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M phase. Formation of pre-RCs and their maintenance during G1 depend on the synthesis and activity of an unstable protein encoded by CDC6. We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and Clb6/Cdk1 in late G1. The latter corresponds to a "point of no return," after which Cdc6 synthesis can no longer promote DNA replication. Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G2 and M phase cells without inducing re-replication. Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing. Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once C1b/Cdk1 kinases have been activated. We show that Cdc6p associates with Clb/Cdk1 kinases from late G1 until late anaphase, which might be important for inhibiting pre-RC assembly during S, G2, and M phases. Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.

Anaphase↗
PubMed1999

Two regions of simian virus 40 T antigen determine cooperativity of double-hexamer assembly on the viral origin of DNA replication and promote hexamer interactions during bidirectional origin DNA unwinding.

Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in which this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA structure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has remarkably similar properties, although these proteins are phosphorylated on threonine 124, as we show here. By comparing the DNA binding properties of the T124A and class 4 mutant proteins with those of the wild type, we demonstrate that mutant double hexamers bind to viral origin DNA with reduced cooperativity. We report that T124A T-antigen subunits impair the ability of double hexamers containing the wild-type protein to unwind viral origin DNA, suggesting that interactions between hexamers are also required for unwinding. Moreover, the T124A and class 4 mutant T antigens display dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding. We speculate that one surface of the DNA binding domain in each subunit of one hexamer may form a docking site that can interact with each subunit in the other hexamer, either directly with the N-terminal phosphorylated region or with another region that is regulated by phosphorylation.

Antigens, Polyomavirus Transforming↗
PubMed1992

EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication.

The plasmid origin of DNA replication of Epstein-Barr virus, oriP, is replicated once per cell division, employing cellular replication machinery and only one viral protein. To understand how replication from this origin is initiated and regulated, we purified this viral protein, EBNA1. EBNA1 was expressed in CV-1p cells by using an infectious simian virus 40 vector containing the EBNA1 gene. It was purified in two chromatographic steps to apparent homogeneity. The purified protein is capable of supporting transcription of the luciferase gene from a reporter plasmid carrying the FR enhancer element to which EBNA1 binds. EBNA1 does not have oriP-dependent ATPase activity, indicating that it does not carry out an energy-dependent step in the initiation of DNA replication. However, EBNA1 does mediate an association between the two elements of oriP. We measured this association by binding one of the elements, the enhancer element, to a solid matrix and measuring retention by this element of the other one, the initiator element, in the presence of EBNA1. This retention is specific for DNA fragments containing EBNA1-binding sites. EBNA1 thus can link the two elements of the origin, providing a locally high concentration of EBNA1 at the site of initiation of DNA replication. We propose that this association is important either (i) to affect DNA structure to allow a cellular helicase to initiate DNA strand separation or (ii) to bind replication proteins to bring them to the origin of replication.

Adenosine Triphosphatases↗
PubMed1995

p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner.

The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that is necessary for the G1 arrest of many cell types. Consistent with its role as a cell cycle checkpoint factor, p53 has been shown to be capable of both transcriptional activation and repression. Here we show a new potential role for p53 as a DNA-binding-dependent regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the late side of the polyomavirus origin were used in in vitro replication assays. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a mutant version of the RGC site was not affected by p53. Several tumor-derived mutant p53 proteins were unable to inhibit replication of the construct with wild-type RGC sites. Additionally, the transactivator GAL4-VP16 was unable to inhibit replication of a construct containing GAL4 binding sites adjacent to the polyomavirus origin. We also show that the inhibition by p53 can occur from sites cloned as far as 600 bp from the origin. Preincubation experiments suggest that p53 inhibits replication at a step mediated by ATP, possibly by inhibiting the binding of polyomavirus T antigen to the core origin. The presence of an endogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition.

Amino Acid Sequence↗
PubMed1990

Chromatin assembly on replicating DNA in vitro.

Replicating single-stranded DNA is preferentially assembled into chromatin in Xenopus egg extracts relative to non-replicating double-stranded DNA. We have examined the molecular basis of this phenomenon. Single-stranded DNA itself is not a favored template for nucleosome assembly in comparison to double-stranded DNA. Complementary strand synthesis is required for the rapid assembly of nucleosomes. We present evidence that the assembly of chromatin on replicating DNA is a two step phenomenon. The first step involves the replication of DNA and the assembly of an intermediate structure, the second step involves the sequestration of histones H2A/H2B onto DNA. Histones H2A/H2B are preferentially sequestered onto replicated DNA in comparison to non-replicated DNA incubated in the extract.

Animals↗
PubMed1996

Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.

Amino Acid Sequence↗
PubMed1977

The units of DNA replication in the mammalian chromosomes: evidence for a large size of replication units.

The replication of chromosomal DNA in human and Chinese hamster cell populations has been studied by means of the DNA fiber autoradiography. It was found that the rate of DNA replication for one fork in human cells varies from 0.2 to 0.9 micron/min, the average being 0.6 micron/min. In the Chinese hamster cells the rate DNA replication is greater, varying from 0.3 to 1.2 micron/min, the average being 0.8 micron/min. There are no clusters containing a great number of replication units in human and Chinese hamster cells. Sequences consisting of two or three replicons which belong to single DNA molecule have been observed, but their frequency was relatively low. The distances between the initiation points in such sequences of replicons vary from 40 to 280 micron, the average value being 130 micron. This value represents the minimum size of the replication units which have completed the DNA synthesis within 3 h of the S-period. The DNA synthesis in most replication units fails to be accomplished within the three hours of labelling. The process can be completed only in the fragments of DNA molecules of 40 to 200 micron (the average value being 100 micron) in human cells, whereas in the Chinese hamster cells the fragments of 40 to 250 micron (the average being about 140 micron) are completely replicated. Provided that the replicaton is bidirectional the complete replicons are supposed to contain two such fragments. Consequently, the greater part of replication units in mammalian cells covers the pieces of a few hundred microns in DNA molecules. The relation between replication process at the DNA molecules level and that at the metaphase chromosome level is discussed.

Autoradiography↗
PubMed2005

Compartmentalization of prokaryotic DNA replication.

It becomes now apparent that prokaryotic DNA replication takes place at specific intracellular locations. Early studies indicated that chromosomal DNA replication, as well as plasmid and viral DNA replication, occurs in close association with the bacterial membrane. Moreover, over the last several years, it has been shown that some replication proteins and specific DNA sequences are localized to particular subcellular regions in bacteria, supporting the existence of replication compartments. Although the mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown, the docking of replication factors to large organizing structures may be important for the assembly of active replication complexes. In this article, we review the current state of this subject in two bacterial species, Escherichia coli and Bacillus subtilis, focusing our attention in both chromosomal and extrachromosomal DNA replication. A comparison with eukaryotic systems is also presented.

Bacillus subtilis↗
PubMed1998

DNA replication machinery of the mammalian cell.

The process of DNA replication in mammalian cells is highly complex and has several unique features that distinguish it from simpler prokaryotic systems. The study of mammalian DNA replication lagged behind that of prokaryotes for many years. This was because of the lack of a reliable and efficient mammalian cell-based in vitro DNA replication system. In 1984, the first mammalian-based DNA replication system that initiated DNA synthesis successfully in vitro was developed. The employment of the mammalian in vitro DNA replication system has led to the identification of several DNA replication proteins. This article describes the current knowledge regarding the proteins mediating mammalian DNA replication, as well as how they are proposed to function during DNA synthesis. There is also a discussion of the role the mammalian cell nuclear architecture plays in DNA replication. The evidence for the existence of an organized DNA replication machine in mammalian cells is also presented.

Animals↗
PubMedDate not supplied

p21CDKN1A does not interfere with loading of PCNA at DNA replication sites, but inhibits subsequent binding of DNA polymerase delta at the G1/S phase transition.

The ability of the cyclin-dependent kinase (CDK) inhibitor p21CDKN1A to interact with PCNA recruited to DNA replication sites was investigated to elucidate the relevance of this interaction in cell cycle arrest. To this end, expression of p21 protein fused to green fluorescent protein (GFP) was induced in HeLa cells. G1 phase cell cycle arrest induced by p21GFP occurred also at the G1/S transition, as shown by cyclin A immunostaining of GFP-positive cells. Confocal microscopy analysis and co-immunoprecipitation studies showed that p21GFP co-localized and interacted with chromatin-bound PCNA and CDK2. GFP-p21 mutant forms unable to bind to PCNA (p21PCNA-) or CDK (p21CDK-) induced cell cycle arrest, although immunoprecipitation experiments showed these mutants to be unstable. Expression of HA-tagged p21wt or mutant proteins confirmed the ability of both mutants to arrest cell cycle. p21(wt)HA and p21CDK-HA, but not p21PCNA-, co-localized and co-immunoprecipitated with chromatin-bound PCNA. Association of p21 to chromatin-bound PCNA resulted in the loss of interaction with the p125 catalytic subunit of DNA polymerase delta (pol delta). These results suggest that in vivo p21 does not interfere with loading of PCNA at DNA replication sites, but prevents, or displaces subsequent binding of pol delta to PCNA at the G1/S phase transition.

CDC2-CDC28 Kinases↗
PubMed1997

Identification of sequence requirement for the origin of DNA replication in human papillomavirus type 18.

DNA replication of human papillomavirus type 18 is dependent on viral proteins E1 and E2 and the subsequent interaction of these proteins with the viral origin of replication. Using a site-directed mutagenesis analysis, we examined the sequence requirement for the DNA replication of the human papillomavirus type 18. We showed that both the E1BS palindrome and E2BS are the major determinants of the HPV replication efficiency. In particular, abolishing E2 binding sites demonstrated that E2BS makes a significant contribution towards HPV-18 DNA replication. Each part of the 18-bp inverted repeat sequence of the E1BS motif showed a clear functional difference between two regions: nt 13-21 (3' half segment) is evidently more important for replication than nt 4-12 (5' half segment). Besides E1BS and E2BS, cis-acting elements such as the poly-A6 track, perhaps the YY1 binding site, and the TATA box sequence within the origin region exhibited some contributions to optimum replication. In addition, inserting an enhancer region to the minimum origin DNA derivatives increased replication approximately 2-fold compared with the wild type levels and showed some compensational effects on loss of the cis-element within the HPV-18 minimum origin, suggesting that an enhancer region is required for efficient replication of the papillomavirus origin. These results suggest that the formation of an E1-E2-ori complex is important for replication, and other sequences near the E1 and E2 binding sites assist E1-E2-ori-mediated DNA replication in vivo.

Base Sequence↗
PubMed2002

Retinoblastoma tumor suppressor targets dNTP metabolism to regulate DNA replication.

The retinoblastoma tumor suppressor, RB, is a negative regulator of the cell cycle that is inactivated in the majority of human tumors. Cell cycle inhibition elicited by RB has been attributed to the attenuation of CDK2 activity. Although ectopic cyclins partially overcome RB-mediated S-phase arrest at the replication fork, DNA replication remains inhibited and cells fail to progress to G(2) phase. These data suggest that RB regulates an additional execution point in S phase. We observed that constitutively active RB attenuates the expression of specific dNTP synthetic enzymes: dihydrofolate reductase, ribonucleotide reductase (RNR) subunits R1/R2, and thymidylate synthase (TS). Activation of endogenous RB and related proteins by p16ink4a yielded similar effects on enzyme expression. Conversely, targeted disruption of RB resulted in increased metabolic protein levels (dihydrofolate reductase, TS, RNR-R2) and conferred resistance to the effect of TS or RNR inhibitors that diminish available dNTPs. Analysis of dNTP pools during RB-mediated cell cycle arrest revealed significant depletion, concurrent with the loss of TS and RNR protein. Importantly, the effect of active RB on cell cycle position and available dNTPs was comparable to that observed with specific antimetabolites. Together, these results show that RB-mediated transcriptional repression attenuates available dNTP pools to control S-phase progression. Thus, RB employs both canonical cyclin-dependent kinase/cyclin regulation and metabolic regulation as a means to limit proliferation, underscoring its potency in tumor suppression.

Adenoviridae↗