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PubMed1992

On the nature of origins of DNA replication in eukaryotes.

Chromosomal origins of DNA replication in higher eukaryotes differ significantly from those of E. coli (oriC) and the tumor virus, SV40 (ori sequence). Initiation events appear to occur throughout broad zones rather than at specific origin sequences. Analysis of four chromosomal origin regions reveals that they share common modular sequence elements. These include DNA unwinding elements, pyrimidine tracts that may serve as strong DNA polymerase-primase start sites, scaffold associated regions, transcriptional regulatory sequences, and, possibly, initiator protein binding sites and inherently destabilized regions. Based on the novel organization of chromosomal origin regions, we propose a model for initiation of DNA replication in higher eukaryotes. Unwinding of duplex DNA during initiation may be uncoupled, both temporally and spatially, from DNA synthesis, resulting in transient single-stranded intermediates that function in lieu of conventional replication forks during chromosomal DNA replication. DNA synthesis begins subsequently at multiple sites within the unwound regions rather than at specific origin sequences.

Animals↗
PubMed1994

Strain-specific mobilization and amplification of a transgenic defective-interfering DNA of the geminivirus beet curly top virus.

Transgenic Nicotiana benthamiana plants have been constructed which bear integrated, tandemly repeated copies of a beet curly top virus (BCTV) defective-interfering (DI) DNA derived from the Logan strain. Transgenic DI-DNA plant lines challenge-inoculated with BCTV-Logan exhibited delayed and attenuated symptoms compared to nontransgenic plants. Infection of transgenic plants with the Logan strain resulted in the mobilization of the integrated DI-DNA sequence, which was subsequently amplified as an episome. The accumulation of Logan helper virus DNA forms was reduced in transgenic plants, relative to nontransgenic plants. In contrast, no delay or attenuation of symptoms was observed for transgenic plants challenge-inoculated with the BCTV strains CFH and Worland. Infection by the CFH and Worland strains did not result in mobilization or amplification of the integrated Logan DI-DNA sequence, and no consistent differences in the accumulation of CFH or Worland genomic viral DNA forms were observed among transgenic and nontransgenic plants. These results, and a comparison of putative DNA replication origin sequences, suggest that BCTV strains display specificity with respect to recognition of heterologous DNA replication origin cis-elements.

Base Sequence↗
PubMed1989

Sequence organization in regulatory regions of DNA of minute virus of mice.

Analysis of the nucleotide sequence of minute virus of mice (MVM) DNA indicates that the DNA termini contain clusters of potential DNA regulatory elements and that there are repetitive DNA elements highly reiterated throughout the entire genome, which may also have a role in DNA function. The left end of MVM DNA, which contains the promoter for the nonstructural genes, has a cluster of DNA elements that includes homologies to the polyoma virus enhancer, three copies of an E1A-inducible transcription factor (ATF) binding site, and a potential Z-DNA element. The MVM right end, which contains the origin of DNA replication, has a cluster of DNA elements that includes several homologies to the polyoma virus replication origin and a potential Z-DNA element. In addition, oligonucleotide frequency analysis indicates the presence of highly recurring sequence elements throughout the entire MVM genome that may be involved in regulation. This computer-aided analysis suggests similarities and significant differences in regulatory sequence organization between MVM and polyoma virus, and identifies specific DNA elements for future genetic characterization.

Activating Transcription Factors↗
PubMed1975

S-phase dependent forms of DNA - nuclear membrane complexes in HeLa cells.

DNA - nuclear membrane complexes were isolated from HeLa cells and examined by either zone sedimentation analysis or isopycnic centrifugation in sucrose/CsCl gradients. The data suggest that the complexes formed during the first 10 min of the S-phase remain as stable structures throughout the cell cycle. Other DNA - nuclear membrane complexes are formed at later times during replication. These later complexes appear as multiple species and the association of DNA and the nuclear membrane seems to be of a transient nature. Together, these results suggest that both the replicative origins and the replication points of the DNA are associated with the nuclear membrane. Although the complexes formed at the start of the S-phase and at later times during the S-phase appear to differ, these differences may provide them with the needed properties to serve as spatial organizers for the temporal regulation of DNA replication.

Binding Sites↗
PubMed1987

Multiple mechanisms for initiation of ColE1 DNA replication: DNA synthesis in the presence and absence of ribonuclease H.

A transcript (RNA II) of plasmid ColE1 that hybridizes with the template DNA is cleaved by RNAase H and used as a primer by DNA polymerase I. However, the plasmid can replicate in bacteria lacking both enzymes, apparently using a different mechanism of initiation of replication. Here we report in vivo and in vitro studies on initiation of DNA replication in the presence or absence of either or both enzymes. Hybridization of RNA II with the template DNA is always required for initiation. Hybridized RNA II is cleaved by RNAase H to form a primer or used as a primer without cleavage by RNAase H. Hybridization also creates a single-stranded region on the nontranscribed strand that can serve as a template for synthesis of the lagging strand in a reaction that does not require DNA polymerase I. Lagging strand synthesis terminates 17 nucleotides upstream of the normal replication origin, forcing unidirectional replication.

Bacteriocin Plasmids↗
PubMed1998

Identification, expression, and characterization of the pseudorabies virus DNA-binding protein gene and gene product.

The pseudorabies virus (PRV) gene encoding a DNA-binding protein (DBP) was first identified in this study. The DBP gene has an open reading frame of 3531 nucleotides, capable of coding a 1177-amino-acid polypeptide of 125 kDa. The deduced DBP exhibits a conserved zinc-binding motif and a conserved DNA-binding region, suggesting the similar DNA-binding mechanism occurs among alphaherpesviral DBP homologs. To further identify the biochemical properties of PRV DBP, this protein was expressed in Escherichia coli by using a pET expression vector and purified to homogeneity. The PRV DBP binds cooperatively and preferentially to single-stranded DNA with no significant base preference, judged by agarose gel electrophoresis and competitive nitrocellulose filter binding assays. Taken together, these results suggest that PRV DBP may play an important role in PRV DNA replication by binding cooperatively and nonspecifically to single-stranded DNA that is formed during the replication origin unwinding and replication fork movement.

Amino Acid Sequence↗
PubMedDate not supplied

Inequality in mutation rates of the two strands of DNA.

As the mechanisms for replicating the two strands of duplex DNA differ it is, in principle, possible for the mutation rates to differ depending on which strand is being copied. In the absence of selection this would lead to a difference in the measured rate of a particular base substitution, such as T to C, depending on which DNA strand was analysed to determine the rate. Thus a change such as T to C on one DNA strand results from either a direct T-to-C mutation on that strand or an A-to-G mutation on the complementary strand; for the other strand the situation is reversed, and it can be seen that different processes are responsible for the two cases, allowing for asymmetry in substitution rate. We have tested whether such asymmetry indeed occurs by studying equivalent sequences from the beta-globin complexes of six species of primate. Our results reveal an asymmetry in substitution rates consistent with predictions based on strand-inequalities in mutation rates. Our sequence comparisons also allow us to make predictions about the positions of replication origins and the replication error rates of one strand relative to the other.

Animals↗
PubMed2000

Multiple independent horizontal transfers of informational genes from bacteria to plasmids and phages: implications for the origin of bacterial replication machinery.

In contrast to the universality of other central genetic mechanisms, the replication machinery of Bacteria is clearly different from those of Archaea and Eukaryotes. A large number of bacterial genes involved in DNA replication can also be found in plasmids and phages. Based on this, it has been recently proposed that the ancestral bacterial genes were displaced by non-orthologous replication genes from plasmids and phages, which would explain the profound difference between Bacteria and the other domains of life. The alternative hypothesis is that these DNA replication genes have been frequently transferred from bacterial hosts to the genomes of their plasmids and phages. The phylogenetic analysis of the bacterial DNA replication proteins most abundant in databases (replicative helicase DnaB, single-strand binding protein Ssb and topoisomerase TopB) presented here supports the latter hypothesis. Each protein tree shows that sequences from plasmids and phages branch close to their bacterial-specific hosts, suggesting multiple independent horizontal transfers. Therefore, there is no evidence so far for non-orthologous gene displacement of these genes.

Bacteria↗
PubMed2000

A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: application to interaction screening.

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein-Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the OriP Epstein-Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an OriP-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/Ori-P system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.

Animals↗
PubMed2006

Overexpression of a cytochrome b5 reductase-like protein causes kinetoplast DNA loss in Trypanosoma brucei.

The mitochondrial genome of trypanosomes, termed kinetoplast DNA (kDNA), contains thousands of minicircles and dozens of maxicircles topologically interlocked in a network. To identify proteins involved in network replication, we screened an inducible RNA interference-based genomic library for cells that lose kinetoplast DNA. In one cloned cell line with inducible kinetoplast DNA loss, we found that the RNA interference vector had aberrantly integrated into the genome resulting in overexpression of genes down-stream of the integration site (Motyka, S. A., Zhao, Z., Gull, K., and Englund, P. T. (2004) Mol. Biochem. Parasitol. 134, 163-167). We now report that the relevant overexpressed gene encodes a mitochondrial cytochrome b(5) reductase-like protein. This overexpression caused kDNA loss by oxidation/inactivation of the universal minicircle sequence-binding protein, which normally binds the minicircle replication origin and triggers replication. The rapid loss of maxicircles suggests that the universal minicircle sequence-binding protein might also control maxicircle replication. Several lines of evidence indicate that the cytochrome b(5) reductase-like protein controls the oxidization status of the universal minicircle sequence-binding protein via tryparedoxin, a mitochondrial redox protein. For example, overexpression of mitochondrial tryparedoxin peroxidase, which utilizes tryparedoxin, also caused oxidation of the universal minicircle sequence-binding protein and kDNA loss. Furthermore, the growth defect caused by overexpression of cytochrome b(5) reductase-like protein could be partially rescued by simultaneously overexpressing tryparedoxin.

Animals↗
PubMed1979

Selective association of the chromosome with membrane in a stable L-form of Bacillus subtilis.

A stalbe L-form (Sal-1) of Bacillus subtilis was found to have retained a markedly modified chromosome-membrane association when compared to intact cells. The membrane-deoxyribonucleic acid complex of the L-form was similar to that of its parental strain in quantity and stability. Genetic analysis of the L-form membrane-deoxyribonucleic acid complex revealed enrichment for markers close to the replication origin, but not for internal markers, indicating preferential attachment of the origin of chromosomal replication to the membrane. These results are in close agreement with those found for the parental bacterial form. In contrast, the replication termius region was not preferentially attached to the membrane of the L-form, even though it is enriched in the bacterial form. The association of the chromosome with the membrane at the replication terminus does not appear to be necessary for cell growth and separation, but because the L-form divides aberrantly, it may be one of the factors required for normal deoxyribonucleic acid segregation and septation.

Bacillus subtilis↗
PubMed1985

Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids.

DNA Replication↗
PubMed2006

Chromosome evolution in the Thermotogales: large-scale inversions and strain diversification of CRISPR sequences.

In the present study, the chromosomes of two members of the Thermotogales were compared. A whole-genome alignment of Thermotoga maritima MSB8 and Thermotoga neapolitana NS-E has revealed numerous large-scale DNA rearrangements, most of which are associated with CRISPR DNA repeats and/or tRNA genes. These DNA rearrangements do not include the putative origin of DNA replication but move within the same replichore, i.e., the same replicating half of the chromosome (delimited by the replication origin and terminus). Based on cumulative GC skew analysis, both the T. maritima and T. neapolitana lineages contain one or two major inverted DNA segments. Also, based on PCR amplification and sequence analysis of the DNA joints that are associated with the major rearrangements, the overall chromosome architecture was found to be conserved at most DNA joints for other strains of T. neapolitana. Taken together, the results from this analysis suggest that the observed chromosomal rearrangements in the Thermotogales likely occurred by successive inversions after their divergence from a common ancestor and before strain diversification. Finally, sequence analysis shows that size polymorphisms in the DNA joints associated with CRISPRs can be explained by expansion and possibly contraction of the DNA repeat and spacer unit, providing a tool for discerning the relatedness of strains from different geographic locations.

Base Sequence↗
PubMed2004

The mammalian beta globin origin of DNA replication.

Initiation of DNA replication is a tightly regulated process aimed to insure that the entire genome is replicated at the appropriate time during each cell cycle. In the human beta globin locus, replication initiates from a region between the two genes that encode the adult subunit of hemoglobin (the beta globin initiation region, or IR). Mammalian beta globin loci replicate early during the S phase of the cell cycle in pre erythroid cells, in which the beta-globin locus is present in a euchromatin form. However, in cells that do not express globin and in which the locus is heterochromatic, these same loci replicate during the later stages of S phase. Both early and late replication patterns utilize similar replication initiation regions. These features make the beta globin locus an attractive model for studying the determinants of replication sites and replication timing, as well as the correlation between gene expression and DNA replication. Two genomic domains are essential for initiation of DNA replication within the locus: the initiation region (IR), and a 40 kb region upstream of the globin gene cluster known as the locus control region (LCR). The IR meets the genetic requirements for a chromosomal replicator, since it can initiate DNA replication at ectopic sites. The LCR regulates transcriptional activity and chromatin structure, and may act as a determinant of replication timing. This review will summarize recent findings characterizing the sequence requirements for initiation of DNA replication in mammalian beta globin loci and will discuss the specific influence of the location and the chromosomal environment in regulating DNA replication at the beta globin IR.

Animals↗
PubMed2006

Turning the replication checkpoint on and off.

The replication checkpoint monitors the progress of DNA replication forks during S phase, and delays the firing of later replication origins when active replication forks are stalled due to collisions with damaged or abnormally structured DNA. Key components of the replication checkpoint pathway are the apical protein kinase, ATR, and its downstream target kinase, Chk1. Defects in either ATR or Chk1 function result in loss of DNA replication fidelity and cell viability, even in the absence of extrinsic genotoxic stress. Moreover, several clinically important antitumor agents, such as the camptothecins (CPTs), exert their antitumor effects by interfering with DNA replication, and hence the therapeutic response to these drugs is intimately related to signaling through the replication checkpoint. A recent report from this laboratory adds a new facet to the regulatory mechanisms that control the function and duration of checkpoint signaling through the ATR-Chk1 pathway. The results indicate that replication stress induced by a variety of agents, including CPT and deep hypoxia, triggers the ubiquitin-dependent degradation of the checkpoint kinase Chk1 in both normal and transformed human cells. This review provides an overview of the study's major findings, together with their implications for both replication checkpoint function and tumor responsiveness to CPT and related anticancer drugs.

Checkpoint Kinase 1↗
PubMed1989

Repression of polyoma virus DNA replication by 5'-flanking region of mouse DNA polymerase beta gene containing transcriptional silencer elements.

Dual cis-acting silencer elements are located upstream of the mouse DNA polymerase beta gene (Yamaguchi, M., Hayashi, Y., and Matsukage, A. (1989) J. Biochem. (Tokyo) 105, 79-83). In order to examine possible involvement of transcriptional silencer elements in the regulation of DNA replication, we have utilized a transient replication system of the plasmid DNA carrying replication origin of polyoma virus DNA in mouse MOP8 cells, which is constitutively producing polyoma virus large T-antigen. The polyoma virus origin of DNA replication is composed of three cis-acting genetic elements called alpha, beta, and core, in which alpha and beta elements correspond to enhancer domains. When the 5'-flanking regions of the DNA polymerase beta gene containing silencer elements were placed at the late gene border of alpha element, they effectively repressed the DNA replication. However, when placed at the early gene border of core element, it only marginally repressed the DNA replication. These results suggest that the silencer elements at cis position repress polyoma virus DNA replication by impeding the enhancer function that activates the DNA replication.

Animals↗
PubMed1987

[Effect of exogenous DNAses on various stages of DNA replication in microorganisms].

Effect of DNAase 1 on DNA synthesis and cell division was studied in microorganisms deficient in some stages of DNA replication initiation. The DNA synthesis induced by exogenous DNAase was found to be a replicative origin since it was registered from the "origin" of chromosomal replication under the conditions of initiation of proteins functioning. Stimulation of DNA synthesis in bacterial cells having mutations in DNA B and DNA G genes by DNAase 1 indicates that exogenous DNAases participate in replicative fork during the DNA synthesis.

DNA Replication↗