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At least 613 records · Page 34Linked to original sources
PubMed2002

The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1).

G(1) cell cycle regulators are often mutated in cancer, but how this causes genomic instability is unclear. Here we show that yeast lacking the CDK inhibitor Sic1 initiate DNA replication from fewer origins, have an extended S phase, and inefficiently separate sister chromatids during anaphase. This leads to double-strand breaks (DSBs) in a fraction of sic1 cells as evidenced by the accumulation of Ddc1 foci and a 575-fold increase in gross chromosomal rearrangements. Both S and M phase defects are rescued by delaying S-CDK activation, indicating that Sic1 promotes origin licensing in late G(1) by preventing the untimely activation of CDKs. We propose that precocious CDK activation causes genomic instability by altering the dynamics of S phase, which then hinders normal chromosome segregation.

Cell Cycle Proteins↗
PubMed2005

Multiple replication origins within the inverted repeat region of the Plasmodium falciparum apicoplast genome are differentially activated.

The 35 kb circular genome (plDNA) of the Plasmodium falciparum apicoplast replicates by the bidirectional ori/D-loop mechanism. PlDNA replication was previously shown to initiate within the inverted repeat (IR) region of the apicoplast genome [Williamson DH, Preiser PR, Moore PW, McCready S, Strath M, Wilson RJM (Iain). The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms. Mol Microbiol 2002;45:533-42; Singh D, Chaubey S, Habib S. Replication of the Plasmodium falciparum apicoplast DNA initiates within the inverted repeat region. Mol Biochem Parasitol 2003;126:9-14.] and the presence of at least two ori within each segment of the IR was postulated. Using 5' end-labelled nascent DNA as probe, we now demonstrate the utilization of several putative ori located within the IR for plDNA replication. Quantitation of signals obtained for different segments of the IR as well as determination of the number of molecules emanating from two ori regions by competitive PCR analysis indicated differential strengths of ori during plDNA replication prior to schizogony.

Animals↗
PubMed2004

Defined sequence modules and an architectural element cooperate to promote initiation at an ectopic mammalian chromosomal replication origin.

A small DNA fragment containing the high-frequency initiation region (IR) ori-beta from the hamster dihydrofolate reductase locus functions as an independent replicator in ectopic locations in both hamster and human cells. Conversely, a fragment of the human lamin B2 locus containing the previously mapped IR serves as an independent replicator at ectopic chromosomal sites in hamster cells. At least four defined sequence elements are specifically required for full activity of ectopic ori-beta in hamster cells. These include an AT-rich element, a 4-bp sequence located within the mapped IR, a region of intrinsically bent DNA located between these two elements, and a RIP60 protein binding site adjacent to the bent region. The ori-beta AT-rich element is critical for initiation activity in human, as well as hamster, cells and can be functionally substituted for by an AT-rich region from the human lamin B2 IR that differs in nucleotide sequence and length. Taken together, the results demonstrate that two mammalian replicators can be activated at ectopic sites in chromosomes of another mammal and lead us to speculate that they may share functionally related elements.

Animals↗
PubMed1976

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: Change of chromosome replication origin in synchronized cultures.

We have followed, by deoxyribonucleic acid-deoxyribonucleic acid hybridization, the order of replication of three chromosomal markers during a synchronous round of replication in three strains of Escherichia coli carrying a dnaAts mutation: one strain in which the F-like R factor R.100.1 was established as a plasmid and two strains in which the dnaA mutation was suppressed by the integration of R.100.1 into the chromosome. In the R+ strain at 30C, replication of the plasmid took place simultaneously with the initiation of chromosome replication at the normal origin. In the integratively suppressed Hfr strains, at 42.5 C, chromosome replication was initiated preferentially from the integrated plasmid; little or no initiation occurred at the normal origin. Similar results were obtained for the one strain tested at 30 C. For both Hfr strains at 42.5 C, the data suggest that at least part of the population replicated bidirectionally. This conclusion had been confirmed using an autoradiographic procedure. Both types of experiment indicate a wide variation in the rate of travel of individual replication forks within the population.

Cell Division↗
PubMed2006

Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress.

In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2-7 (Mcm2-7). The number of Mcm2-7 complexes loaded onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2-7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2-7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.

Adenosine Triphosphatases↗
PubMed1998

Active domains of human papillomavirus type 11 E1 protein for origin replication.

Viral proteins E1 and E2 are essential for transient human papillomavirus (HPV) DNA replication. E1 is a multifunctional protein which can bind DNA and complex with E2, has ATPase and helicase activities, and interacts with DNA polymerase alpha-primase. E2 is a transactivator-repressor protein, playing an important role in replication and transcriptional regulation. A series of deletion mutants of HPV-11 E1 were constructed and tested in functional assays to define those domains of HPV-11 E1 which are important for binding to the origin DNA and E2. The domain of HPV-11 E1 involved in binding to the origin was located between aa 186 and 649, and that for binding to E2 was between aa 346 and 649. Since E1 binds to the origin more efficiently in the presence of E2, we also mapped the DNA binding domain of E1 in the presence of E2, and found that when binding was enhanced, the region of E1 involved in binding was similar to that observed with E1 alone. The same deletion mutation constructs of E1 were subcloned into an expression vector for use in transient replication assays to study the effect of the deletions on the replication of the origin DNA in vivo and the data suggest that the C-terminal domain contains important functions for replication.

Binding Sites↗
PubMed1982

Dissection of a replication origin of Xenopus DNA.

A previously cloned 503-base pair (bp) EcoRI segment of genomic DNA from Xenopus laevis selected for enhancement of replication of its vector plasmid was moved to the EcoRI site of pBR322. This plasmid designated pJCC31 and five other clones, which were made by cleaving the 503-bp segment in relation to a dispersed repeated sequence and subcloning, were compared with pBR322 for replication by microinjection into Xenopus eggs. The replication measured by incorporation of a 32P-labeled nucleotide as well as semiconservative segregation and dilution of N6-methyladenine at the EcoRI sites showed pJCC31 to be about 15 times as efficient as pBR322. The next most efficient subclone, pJCC31-2, contains an insert with a complete 320-bp dispersed repeated sequence bracketed by an 8-bp direct repeat. This observation, along with our previous report that repeated sequences of the Alu family in the human genome enhanced replication of the vector plasmid nearly as much as that of the presumptive Xenopus origin, leads to the hypothesis that members of a subset of the short dispersed repeated sequences in vertebrates function as origins for chromosomal replication. Preliminary studies also show that the presumptive Xenopus origin contains a RNA polymerase promoter that increases the transcription of the plasmid when it is microinjected into Xenopus oocytes.

Animals↗
PubMed1997

Mapping replication origins by neutral/neutral two-dimensional gel electrophoresis.

Neutral/neutral two-dimensional gel electrophoresis is a sensitive physical mapping technique that has been successfully used to unambiguously identify replication initiation sites in genomes of widely varying complexity in vivo. The technique exploits the fact that restriction fragments containing different classes of replicative intermediates (single forks, initiation bubbles, or termination structures) migrate to different and characteristic positions in agarose gels. The replication pattern of any region of interest can then be determined by sequential hybridization with appropriate radioactive probes from that region.

Animals↗
PubMed1996

Conditional requirement for sequences distinct from the replication origin during episomal establishment of the BPV1 genome.

Removal from BPV1 DNA of a short segment (nt. 4786-5045) that contains several protein binding sites and is required for efficient replication in short term assays prevents its autonomous maintenance in cell lines established by selection in G418 medium after cotransfer of neo(r). In contrast, transformed cell lines established from foci, which express the viral genes at higher levels, maintain extrachromosomal copies of the deleted DNA. Two modes of maintenance of the viral genome are thus distinguished by their requirement for sequences in this region.

Animals↗
PubMed1985

Nucleotide sequence analysis of DNA replication origins of the small Bacillus bacteriophages: evolutionary relationships.

The ends of the small Bacillus phage genomes serve as origins and termini of their DNA replication. We have determined nucleotide sequences at the termini of four different phage DNAs and compared them with those of phi 29 DNA which has been described previously. A high degree of homology was found at the extreme ends of DNAs from phi 29, phi 15 (group A), M2Y and Nf (group B). 17 bp at the far left of the DNAs are identical. A highly conserved dodecanucleotide sequence, CCATTTCCCCAT, was also found in the righthand terminus of all these phage DNAs, at positions 27-38 from the end. Nucleotide sequences of phage GA-1 are not very similar to those of the other phages. Examination of the 5'-terminal and 3'-terminal sequences of all the phages suggests that stable 'panhandle' structures are unlikely to be formed via base pairing of both ends. However, thermodynamically more stable panhandle structures might be formed by displaced single-stranded DNA, although this requires rather large loops.

Bacillus subtilis↗
PubMed1989

Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus.

EBNA1 activates the EBV plasmid maintenance sequence oriP by binding to its two essential regions. One region is a family of 30-base-pair (bp) repeats and is activated by EBNA1 to act as a transcriptional enhancer. The other region contains a 65-bp dyad symmetry and lacks enhancer function. To explore the functional differences between the two regions, we determined oriP activities as functions of the number of 30-bp repeats and compared them with activities determined when tandem copies of the dyad symmetry region were used to replace the 30-bp repeats. Three conclusions have been drawn. (i) Activation of the 30-bp repeats by EBNA1 to enhance transcription or to permit plasmid maintenance is a highly cooperative process involving at least six or seven 30-bp repeats for full activity. (ii) Tandem copies of the dyad symmetry region cooperatively enhance transcription but are less effective than 30-bp repeats providing a similar number of EBNA1-binding sites. (iii) Tandem copies of the dyad symmetry region alone cooperatively activate replication, suggesting that the region contains the actual origin of replication. We also report that while rodent-derived cell lines do not support replication of EBV-derived plasmids they do permit EBNA1-dependent enhancer activity. EBV plasmid replication thus requires the interaction of EBNA1 or oriP with a host factor that is not required for enhancement of transcription.

Antigens, Viral↗
PubMed1983

Complexes of Escherichia coli primase with the replication origin of G4 phage DNA.

Escherichia coli primase (dnaG protein), an essential DNA replication enzyme, synthesizes a primer at the unique origin sequence of the single-stranded circular phage G4 DNA (Rowen, L., and Kornberg, A. (1978) J. Biol. Chem. 253, 758-764). Kinetic analyses suggest that for each DNA molecule at least two primase molecules participate in the reaction. Binding of 3H-labeled primase is specific for the G4 complementary strand origin region and is saturated at approximately 2 primase molecules/DNA circle. Such complexes, isolated by gel filtration, function in the absence of additional primase to convert the phage DNA to the duplex form. Although the primase-DNA complex is stable to refiltration, the DNA-bound enzyme can dissociate and reattach to function at the origin sequence of another G4 DNA circle. An antibody to primase blocks the action of primase in the free form or within a DNA complex and even interferes with extension of the primer by DNA polymerase III holoenzyme. These kinetic and binding studies of G4 priming, the least complicated of the primase systems, suggest that 2 primase molecules form a complex at the origin region and remain bound even after transcribing a sequence to prime DNA replication.

Coliphages↗
PubMed1998

Transformation abrogates an early G1-phase arrest point required for specification of the Chinese hamster DHFR replication origin.

The origin decision point (ODP) was originally identified as a distinct point during G1-phase when Chinese hamster ovary (CHO) cell nuclei experience a transition that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. Passage of cells through the ODP requires a mitogen-independent protein kinase that is activated prior to restriction point control. Here we show that inhibition of an early G1-phase protein kinase pathway by the addition of 2-aminopurine (2-AP) prior to the ODP arrests CHO cells in G1-phase. Transformation with simian virus 40 (SV40) abrogated this arrest point, resulting in the entry of cultured cells into S-phase in the presence of 2-AP and a disruption of the normal pattern of initiation sites at the DHFR locus. Cells treated with 2-AP after the ODP initiated replication specifically within the DHFR origin locus. Transient exposure of transformed cells to 2-AP during the ODP transition also disrupted origin choice, whereas non-transformed cells arrested in G1-phase and then passed through a delayed ODP after removal of 2-AP from the medium. We conclude that mammalian cells have many potential sites at which they can initiate replication. Normally, events occurring during the early G1-phase ODP transition determine which of these sites will be the preferred initiation site. However, if chromatin is exposed to S-phase-promoting factors prior to this transition, mammalian cells, like Xenopus and Drosophila embryos, can initiate replication without origin specification.

2-Aminopurine↗
PubMed2006

Prediction and functional analysis of the replication origin of the linear plasmid pSCL2 in Streptomyces clavuligerus.

pSCL2 (120 kb), one of the linear plasmids found in Streptomyces clavuligerus NRRL3585, was isolated and partially sequenced. Computational analysis of the central region of pSCL2 revealed the presence of two open reading frames that appear to encode proteins highly homologous to RepL1 and RepL2, replication proteins from pSLA2-L, the large linear plasmid in Streptomyces rochei. The S. clavuligerus open reading frames were designated repC1 and repC2, encoding the proteins RepC1 (150 amino acids) and RepC2 (102 amino acids), respectively. The RepC and RepL proteins have identical translation features and very similar predicted secondary and tertiary structures. Functional analysis confirmed that RepC1 is essential for replication initiation of pSCL2, whereas RepC2 is dispensable but may play a role in copy number control. The RepC and RepL proteins do not show similarity to any other bacterial plasmid replication proteins. Three regions of DNA sequence, Box 1 (1050-850 bp), Box 2 (723-606 bp), and Box 3 (224-168 bp), located upstream of repC1, were also shown to be essential or very important for replication of pSCL2.

Bacterial Proteins↗