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At least 1,711 records · Page 95Linked to original sources
PubMed2002

The role of the carboxyterminal domain of RNA polymerase II in regulating origins of DNA replication in Saccharomyces cerevisiae.

MCM (minichromosome maintenance) proteins function as a replication licensing factor (RLF-M), which contributes to limiting initiation of DNA replication to once per cell cycle. In the present study we show that a truncation of the pol II CTD in a S. cerevisiae strain harboring a mutation in mcm5 partially reverses its ts phenotype and improves maintenance of CEN/ARS minichromosomes. We correlate this phenotype to effects on DNA replication rather than to effects on transcription or specific gene expression. We also demonstrate that a similar truncation of the CTD reduces minichromosome stability and impairs stimulation of DNA replication by trans-activators and that tethering of recombinant pol II CTD to an origin of replication has a significant stimulatory effect on minichromosome stability. Furthermore, we show that pol II is recruited to ARS1. We propose that in S. cerevisiae a mechanism of coordinating pol II transcription and DNA replication is mediated by the CTD of pol II.

Cell Cycle Proteins↗
PubMed2004

Where does bacterial replication start? Rules for predicting the oriC region.

Three methods, based on DNA asymmetry, the distribution of DnaA boxes and dnaA gene location, were applied to identify the putative replication origins in 120 chromosomes. The chromosomes were classified according to the agreement of these methods and the applicability of these methods was evaluated. DNA asymmetry is the most universal method of putative oriC identification in bacterial chromosomes, but it should be applied together with other methods to achieve better prediction. The three methods identify the same region as a putative origin in all Bacilli and Clostridia, many Actinobacteria and gamma Proteobacteria. The organization of clusters of DnaA boxes was analysed in detail. For 76 chromosomes, a DNA fragment containing multiple DnaA boxes was identified as a putative origin region. Most bacterial chromosomes exhibit an overrepresentation of DnaA boxes; many of them contain at least two clusters of DnaA boxes in the vicinity of the oriC region. The additional clusters of DnaA boxes are probably involved in controlling replication initiation. Surprisingly, the characteristic features of the initiation of replication, i.e. a cluster of DnaA boxes, a dnaA gene and a switch in asymmetry, were not found in some of the analysed chromosomes, particularly those of obligatory intracellular parasites or endosymbionts. This is presumably connected with many mechanisms disturbing DNA asymmetry, translocation or disappearance of the dnaA gene and decay of the Escherichia coli perfect DnaA box pattern.

Bacteria↗
PubMed1995

Boundaries of the pSC101 minimal replicon are conditional.

The DNA segment essential for plasmid replication commonly is referred to as the core or minimal replicon. We report here that host and plasmid genes and sites external to the core replicon of plasmid pSC101 determine the boundaries and competence of the replicon and also the efficiency of partitioning. Missense mutations in the plasmid-encoded RepA protein or mutation of the Escherichia coli topoisomerase I gene enable autonomous replication of a 310-bp pSC101 DNA fragment that contains only the actual replication origin plus binding sites for RepA and the host-encoded DnaA protein. However, in the absence of a repA or topA mutation, the DNA-bending protein integration host factor (IHF) and either of two cis-acting elements are required. One of these, the partitioning (par) locus, is known to promote negative DNA supercoiling; our data suggest that the effects of the other element, the inverted repeat (IR) sequences that overlap the repA promoter, are mediated through the IR's ability to bind RepA. The concentrations of RepA and DnaA, which interact with each other and with plasmid DNA in the origin region (T. T. Stenzel, T. MacAllister, and D. Bastia, Genes Dev. 5:1453-1463, 1991), also affect both replication and partitioning. Our results, which indicate that the sequence requirements for replication of pSC101 are conditional rather than absolute, compel reassessment of the definition of a core replicon. Additionally, they provide further evidence that the origin region RepA-DnaA-DNA complex initiating replication of pSC101 also mediates the partitioning of pSC101 plasmids at cell division.

Bacterial Proteins↗
PubMed2006

Epstein-Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments.

Epstein-Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain-enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.

Cell Line, Tumor↗
PubMed2001

An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint.

Checkpoint controls coordinate entry into mitosis with the completion of DNA replication. Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response. However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA. In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression. Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint. In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.

Cell Cycle Proteins↗
PubMed1997

Genes involved in conjugative DNA processing of plasmid R6K.

The conjugative transfer region of the IncX plasmid R6K (TRA(x)) was analysed by transposon mutagenesis and DNA sequencing. Tn5tac1 insertional mutations localized TRA(x) to a 14.8 kb segment containing the alpha origin of transfer (oriT alpha), genes involved in conjugative DNA-processing (Dtr(x)) and genes involved in pilus synthesis and assembly (Mpf(x)). A second functional oriT, oriTbeta, was located at a distance of 5.3 kb from oriT alpha and was outside TRA(x). Mpf(x) occupied a segment of 10kb, as judged by the location of insertions conferring resistance to infection by the X pilus-specific phage X-2. At both sides of Mpf(x) there were insertions that were Tra but X-2 sensitive, suggesting that the mutations were in Dtr(x). This region was sequenced and three genes were identified: taxA, taxB, and taxC. The overall organization was oriT alpha-taxA-taxC-Mpf(x)-taxB. taxC coded for a oriT-relaxase that belongs to the VirD2 family. taxA coded for a protein of 181 amino acids that showed similarity to TraY of F-like plasmids and to the Arc-repressor superfamily. TaxB showed similarity to TraG-like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA-transport apparatus. TaxA and TaxC are required for oriT nicking in vivo. The nicking reaction was mistakenly assumed by Flashner et al. (1996) to represent a feature of the vegetative replication origins. However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K. Conversely, protein pi, which is absolutely required for replication of R6K, was not required for conjugative transfer. In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation. Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein pi for conjugation.

Amino Acid Sequence↗
PubMed1996

A dnaA box can functionally substitute for the priming signals in the oriV of the broad host-range plasmid RSF1010.

The initiation of replication from oriV RSF1010, the replication origin of the broad host-range plasmid RSF1010, depends on RepA (helicase), RepB' (primase), and RepC (initiator protein), encoded by RSF1010 itself, while this initiation event in E. coli is independent of dnaA, dnaB, dnaC, and dnaG [Scherzinger et al. (1984) Proc. Natl. Acad. Sci. USA 81, 654-658; Scholz et al. (1985) in: Plasmids in Bacteria, pp. 243-259, Plenum, New York; Haring and Scherzinger (1989) in: Promiscuous Plasmids of Gram-negative Bacteria, pp. 95-124, Academic Press, London; Scherzinger et al. (1991) Nucl. Acids Res. 19, 1203-1211]. We showed in this work that a newly constructed origin consisting of an oriV RSF1010 and a DnaA protein binding site, the dnaA box, inserted near oriV RSF1010 (oriV RSF1010-dnaA box) could function without RepB' primase, but required RepA and RepC. This oriV RsF1010-dnaA box could not replicate in a dnaA46 strain in which only RepA and RepC were supplied, even at a permissive temperature. These results indicate that an inserted dnaA box can functionally substitute for the RSF1010-specific ssi signals, the RepB' dependent priming signals in oriV RSF1010, and can direct a priming pathway different from the RSF1010-specific one, but related to DnaA protein.

Bacterial Proteins↗
PubMed2000

Replication-induced transcription of an autorepressed gene: the replication initiator gene of plasmid P1.

The replication origin of plasmid P1 contains an array of five repeats (iterons) that bind the plasmid-encoded initiator RepA. Within the array lies the repA promoter, which becomes largely repressed on RepA binding (autorepression). One might expect that extra iterons produced on plasmid replication would titrate RepA and release the repression. The promoter, however, is induced poorly by extra iterons. The P1 copy number is reduced by extra iterons in the presence of the autorepressed repA gene but not when additional RepA is provided from constitutive sources. It has been proposed that the iteron-bound RepA couples with the promoter-bound RepA and thereby maintains repression. Although not the product of replication, we find that the act of replication itself can renew RepA synthesis. Replication apparently cleans the promoter of bound RepA and provides a window of opportunity for repA transcription. We propose that replication-induced transcription is required to ensure initiator availability in a system that is induced poorly when challenged with additional initiator binding sites.

DNA Helicases↗
PubMed1996

The presence of tandem repeats and the initiation of replication in rabbit mitochondrial DNA.

The non-coding region of rabbit mitochondrial DNA (mtDNA) exhibits two sets of tandem repeats between conserved sequence block 1 (CSB1) and the tRNA Phe gene. Both repeated sequences, short repeated (SR) and long repeats (LR), which contain 20 and 153 nucleotides, respectively, are involved in the generation of a high degree of mitochondrial heteroplasmy. Due to the location of these sequences in the regulatory region and their properties in terms of variable conformations, they could affect the initiation of replication of the heavy-strand DNA (H-strand DNA) and subsequently would influence the efficiency of mtDNA replication. The extremities of the displacement loop (D-loop) DNA strand and the 5' ends of RNA primers initiating the H-strand DNA synthesis were characterized in individual rabbits. Mapping at the nucleotide level of the 5' and 3' ends of the D-loop DNA strands indicates that both extremities are heterogeneous. The H-strand replication origin OH is located close to the conserved sequence block CSB1 as in other mammals. In all of the individuals studied so far, DNA molecules with a 5' end 1-2 nucleotides downstream of CSB1 were always present. As H-strand DNA replication is believed to be primed by RNA transcribed from the light-strand promoter (LSP), RNA mapping was carried out to identify the 5' end of H-strand RNA. Neighbouring initiation sites were identified at the nucleotide level in an (A+T)-rich region at nucleotide 1841 and in a stretch of cytosine residues at nucleotides 1849-1852, which are located at the beginning of the first long repeat. A detailed RNA analysis indicates that H-strand RNA molecules are initiated in each long repeat. The amplification of the regulatory region has produced multiple initiation transcription sites and a family of RNA primers of various lengths. These variations in length and the ensuing secondary structures are not critical for their potential function as H-strand DNA replication primers.

Animals↗
PubMed1985

Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens.

A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.

Animals↗
PubMed1996

Essential region for self-replication of Coryneform bacteria plasmid pXZ10145.

A pTSK series of recombinant plasmids were constructed by cloning DNA fragments of pXZ10145 or its deleted deriviate pATN65 into plasmid vector pACYC177 of E. coli. Experiment results of Coryneform bacteria transformation with these pTSK plasmids allowed us to localize the essential region for self-replication on plasmid pXZ10145. The minimal replication region of the pXZ10145 was located on a 1.2kb Nael-Nrul DNA fragment in which only one open reading frame was found. This ORF was believed to be encoded a trans-acting replication factor. The replication origin (oriV) was locate on a 0.3kb NaeI-SalI fragment which was within the ORF region.

Actinomycetales↗
PubMed2006

ORC is necessary at the interphase-to-mitosis transition to recruit cdc2 kinase and disassemble RPA foci.

The origin-recognition complex (ORC) has an essential role in defining DNA replication origins and in chromosome segregation. Recent studies in Drosophila orc2 mutants, and in human cells depleted of ORC2, have suggested that this factor is also implicated in mitotic chromosome assembly. We asked whether ORC was required for M phase chromosome assembly independently of its function in DNA replication. We performed depletion assays and reconstitution experiments in Xenopus egg extracts, in conditions of M phase chromosome assembly coupled or uncoupled from DNA replication. We show that, although ORC is dispensable for mitotic chromosome condensation, it is necessary at the interphase-mitosis transition for proper mitotic chromosome assembly to occur in a reaction not strictly dependent on DNA replication. This function involves the recruitment to chromatin of cdc2 kinase and the chromatin disassembly of interphasic replication protein A (RPA) foci. Furthermore, we show that mutations of RPA at the cdc2 kinase site prevents RPA dissociation from chromatin and impairs mitotic chromosome assembly without affecting DNA replication. Our results support the conclusion that in addition to its role in the assembly of prereplication complexes (pre-RCs), at the G1-S transition, ORC is also required for their disassembly at mitotic entry.

Animals↗
PubMed1996

Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution.

Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminvirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.

Amino Acid Sequence↗
PubMed1998

In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): a remnant of bacterial rolling circle and conjugative plasmids?

According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes.

Base Sequence↗
PubMed2006

Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication.

Chromatin is suppressive in nature to cellular enzymes that metabolize DNA, mainly due to the inherent inaccessibility of the DNA template. Despite extensive understanding of the involvement of chromatin-modifying factors in transcription, roles of related activities in DNA replication remain largely elusive. Here, we show that the heterodimeric transcriptional elongation factor FACT (facilitates chromatin transcription) is functionally linked to DNA synthesis. Its involvement in DNA replication is partly mediated by the stable association with the replicative helicase complex, MCM, and further by the coexistence with MCM on replication origin. Furthermore, relying on its nucleosome-reorganizing activity, FACT can facilitate chromatin unwinding by the MCM complex, which is otherwise inert on the nucleosomal template. As a consequence, the physical and functional interaction between FACT and MCM is an important determinant in the proper initiation of DNA replication and S phase in vivo. Together, our findings identify FACT as an integral and conserved component of the endogenous replication machinery, and support a model in which the concerted action of helicase and chromatin-modifying activities promotes chromosome replication.

Chromatin↗
PubMed2004

Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis.

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.

Adenosine Triphosphate↗
PubMed1996

The relationship between sequence-specific termination of DNA replication and transcription.

In Escherichia coli and Bacillus subtilis replication fork arrest occurs in the terminus at sequence-specific sites by the binding of replication terminator proteins to the fork arrest sites. The protein-DNA complex causes polar arrest of the replication forks by inhibiting the activity of the replicative helicases in only one orientation of the terminus with respect to the replication origin. This activity has been named as polar contrahelicase. In this paper we report on a second novel activity of the terminator proteins of E.coli and B.subtilis, namely the ability of the proteins to block RNA chain elongation by several prokaryotic RNA polymerases in a polar mode. The replication terminator proteins ter and RTP of E.coli and B.subtilis respectively, impeded RNA chain elongation catalyzed by T7, SP6 and E.coli RNA polymerases in a polar mode at the replication arrest sites. The RNA chain anti-elongation and the contrahelicase activities were isopolar. Whereas one monomer of ter was necessary and sufficient to block RNA chain elongation, two interacting dimers of RTP were needed to effect the same blockage. The biological significance of the RNA chain anti-elongation activity is manifested in the functional inactivation of a replication arrest site by invasion of RNA chains from outside, and the consequent need to preserve replication arrest activity by restricting the passage of transcription through the terminus-terminator protein complex.

Bacillus subtilis↗