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PubMed1993

Specific chromosomal sites enhancing homologous recombination in Escherichia coli mutants defective in RNase H.

To clone new replication origin(s) activated under RNase H-defective (rnh-) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Kmr DNA fragment and transformed into an rnh- derivative host. From the Kmr transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed "Hot", derived from the E. coli genome. Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection. Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species. While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H. This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.

Chromosomes, Bacterial↗
PubMedDate not supplied

Enzymatic RNA synthesis and RNase P. Evolutionary aspects.

TransferRNA recognition was used as leit-motiv in the illustration of possible links between a hypothetical primordial RNA world and the contemporary DNA world. In an RNA world, 'proto-tRNA' could have functioned as replication origin and as primitive telomere. Possibly, this primitive structure is preserved in a 'universal substrate' for modern tRNA-specific enzymes. The combination of acceptor stem and T arm (plus a linker) was finally revealed as sufficient for the recognition by prokaryotic and eukaryotic RNase P, as well as other tRNA enzymes. In modern life forms, a tRNA-like element in viral RNAs still serves as replication origin, and furthermore, the recognition of similar structures as cryptic promoters is universally conserved for template-dependent RNA polymerases. Another common property of modern polymerases is their high, but clearly limited and condition-dependent substrate specificity. Very likely, also substrate recognition by primitive polymerases was not more stringent, and this lead to the occurrence of mixed nucleic acids as intermediates in the transition of genomic RNA to contemporary genomic DNA.

Biological Evolution↗
PubMed1993

Characterization of the length polymorphism in the A + T-rich region of the Drosophila obscura group species.

In the twelve Drosophila obscura group species studied, belonging to the affinis, obscura, and pseudoobscura subgroups, the mitochondrial DNA length ranges from 15.8 to 17.2 kb. This length polymorphism is mainly due to insertions/deletions in the variable region of the A + T-rich region. In addition, one species (D. tristis) possess a tandem duplication of a 470-bp fragment that contains the replication origin. The same duplication has occurred at least twice in the Drosophila evolutionary history due to the fact that the repetition is analogous to repetitions found in the four species of the D. melanogaster complex. By comparing the nucleotide sequence of the conserved region in D. ambigua, D. obscura, D. yakuba, D. teissieri, and D. virilis, we show the presence of a secondary structure, likely implied in the replication origin, which could favor the generation of this kind of duplications. Finally, we propose that the high A and T content in the variable region of the A + T-rich region favors the formation of less-stable secondary structures, which could explain the generation of minor insertion/deletions found in this region.

Adenine↗
PubMed2004

Functional properties of the herpes simplex virus type I origin-binding protein are controlled by precise interactions with the activated form of the origin of DNA replication.

The herpes simplex virus, type I origin-binding protein, OBP, is a superfamily II DNA helicase encoded by the UL9 gene. OBP binds in a sequence-specific and cooperative way to the viral origin of replication oriS. OBP may unwind partially and introduce a hairpin into the double-stranded origin of replication. The formation of the novel conformation referred to as oriS* also requires the single-stranded DNA-binding protein, ICP8, and ATP hydrolysis. OBP forms a stable complex with oriS*. The hairpin in oriS* provides a site for sequence-specific attachment, and a single-stranded region triggers ATP hydrolysis. Here we use Escherichia coli exonuclease I to map the binding of the C-terminal domain of OBP to the hairpin and the helicase domains to the single-stranded tail. The helicase domains cover a stretch of 23 nucleotides of single-stranded DNA. Using streptavidin-coated magnetic beads, we show that OBP may bind two copies of double-stranded DNA (one biotin-labeled and the other one radioactively labeled) but only one copy of oriS*. It is the length of the single-stranded tail that determines the stoichiometry of OBP.DNA complexes. OBP interacts with the bases of the single-stranded tail, and ATP hydrolysis is triggered by position-specific interactions between OBP and bases in the single-stranded tail of oriS*.

Adenosine Triphosphate↗
PubMed1999

The Drosophila chiffon gene is required for chorion gene amplification, and is related to the yeast Dbf4 regulator of DNA replication and cell cycle.

The Drosophila chorion genes encode the major protein components of the chorion (eggshell) and are arranged in two clusters in the genome. To meet the demand for rapid chorion synthesis, Drosophila ovary follicle cells amplify the chorion gene clusters approximately 80-fold. Amplification proceeds through repeated firing of one or more DNA replication origins located near the center of each gene cluster. Hypomorphic mutant alleles of the chiffon gene cause thin, fragile chorions and female sterility, and were found to eliminate chorion gene amplification. Null alleles of chiffon had the additional phenotypes of rough eyes and thin thoracic bristles: phenotypes often associated with disruption of normal cell cycle. The chiffon locus was cloned by chromosomal walking from the nearby cactus locus. A 6.5 kb transcript was identified and confirmed to be chiffon by sequencing of mutant alleles and by phenotypic rescue with genomic transformation constructs. The protein predicted by translation of the 5.1 kb chiffon ORF contains two domains related to the S. cerevisiae Dbf4 regulator of DNA replication origin firing and cell cycle progression: a 44 residue domain designated CDDN1 (43% identical) and a 41 residue domain designated CDDN2 (12% identical). The CDDN domains were also found in the S. pombe homolog of Dbf4, Dfp1, as well as in the proteins predicted by translation of the Aspergillus nimO gene and specific human and mouse clones. The data suggest a family of eukaryotic proteins related to Dbf4 and involved in initiation of DNA replication.

Amino Acid Sequence↗
PubMed1997

Plasmids in Lactobacillus.

This review describes Lactobacillus plasmids on distribution, structure, function, vector construction, vector stability, application, and prospective. About 38% of species of the genus Lactobacillus were found to contain plasmids with different sizes (from 1.2 to 150 kb) and varied numbers (1 or more). Some Lactobacillus plasmids with small sizes were highly similar to those of single strand plasmids from other Gram-positive bacteria. The extensive sequence homologies of plus origins, replication initiation proteins, minus origins, cointegration sites, and the presence of single strand intermediates supported the fact that these small Lactobacillus plasmids replicate with a rolling-circle replication mechanism. Some Lactobacillus plasmid replicons were of broad host range that could function in other Gram-positive bacteria, and even in Escherichia coli, while replicons of other Gram-positive bacteria also function in Lactobacillus. Although most Lactobacillus plasmids are cryptic, some plasmid-encoded functions have been discovered and applied to vector construction and Lactobacillus identification, detection, and modification.

DNA, Bacterial↗
PubMed1986

Viable viruses with deletions in the left inverted terminal repeat define the adenovirus origin of DNA replication.

A series of human adenovirus type 2 genomes with deletions in the left inverted terminal repeat (ITR) have been constructed. Viral genomes that contained a minimum of 45 base pairs (bp) from the terminus of the genome were fully infectious and gave rise to progeny virus which maintained the deletion. In contrast, genomes containing 36 bp or less from the termini of the genome were not infectious. The boundary of a cis-acting element required for viral replication is therefore between 36 and 45 bp from the adenovirus termini and corresponds to the previously identified viral origin of replication, defined using a transfection assay to measure ori activity in vivo. The growth parameters of viruses with deletions in the left ITR were examined. These deletions had no measurable effect on plaque formation or morphology, viral DNA synthesis or early viral mRNA synthesis. Thus, it appears that DNA sequences in the left ITR, outside the replication origin, are completely dispensable for lytic viral growth in tissue culture cells.

Adenoviruses, Human↗
PubMed1996

Gene targeting in rat embryo fibroblasts promoted by the polyomavirus large T antigen.

We used the recombination-promoting activity of the polyomavirus large T antigen (T-ag) to increase the frequency of gene targeting in rat fibroblasts. We constructed a cell line carrying a functional polyomavirus replication origin and a transformation-defective middle T-ag oncogene. The structure of the locus was such that homologous recombination with the targeting DNA reconstituted a functional transforming gene and converted the cells from the normal to the transformed state. Introduction of the large T-ag with the targeting DNA promoted recombinational events that corrected the mutation in either the target locus or the targeting DNA. The frequency of recombination was not substantially influenced by the extent of homology between the recombining sequences. However, it was reduced when the replication origin was inactivated in the targeting DNA, and was reduced further when the origin was inactivated in the target locus.

Animals↗
PubMed1993

The mechanism of sequence-specific DNA cleavage and strand transfer by phi X174 gene A* protein.

We have examined the biological role and catalytic function of two juxtaposed tyrosyl residues in the bacteriophage phi X174 gene A protein, Tyr-343 and Tyr-347, which have been implicated in the catalysis of sequence-specific DNA strand transfer. Site-directed mutagenesis changing either tyrosine to phenylalanine abolishes phage viability. The biochemical basis of this inviability was studied using purified A* protein containing the carboxyl-terminal 341 amino acids of the A protein, as well as purified A* protein with a Y343F or Y347F mutation. All three proteins can cleave the phi X174 replication origin and perform strand transfer between oligodeoxynucleotides bearing the recognition sequence of the A protein; however, both Tyr-343 and Tyr-347 appear to be required for coordinated DNA strand transfer by a single A* protein molecule. The chirality of a phosphorothioate group at the site of strand transfer in the DNA was found to be retained following the strand-transfer reaction, which argues against transfer of Tyr-343-linked DNA to Tyr-347 on the same protein molecule or vice versa. These results support the current model of gene A protein function in which the two tyrosines of a single protein molecule alternate in catalyzing DNA strand transfer at the viral replication origin.

Bacteriophage phi X 174↗
PubMed1983

A vehicle for DNA transfer and for recovery of transferred genes: lambda Charon phage-pBR322 hybrid.

Recombinant Charon 4A phages accommodating the Herpes simplex virus (HSV-1) thymidine kinase (tk) gene, the ampicillin-resistance (ApR) gene, and the replication origin of pBR322 were constructed. The phage DNA was introduced into mouse Ltk- cells by a free DNA transfer method or phage-mediated DNA transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607]. Analyses of the physical state of the transferred DNA in the recipient cell genome showed that a DNA fragment as long as 12.7 kb was integrated intact into 67% and less than 40% of the Ltk- transformant cells by phage-mediated DNA transfer and by free DNA transfer, respectively. We also developed a new rapid method for recovery of the transferred gene from the Ltk+ cell into Escherichia coli; the method depends on the fact that the recombinant lambda phage carrying the ApR gene and replication origin of pBR322 transduces lambda-lysogenic bacteria to ApR and is maintained as a plasmid. Using this method the HSV-1 tk gene from one Ltk+ transformant was rapidly and successfully recovered without any rearrangement of the target sequence.

Animals↗
PubMed2003

Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development.

Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes. Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases. Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication. In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis. Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase). Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis. Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals. Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells. Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development.

Animals↗
PubMed1989

Transformation of Tetrahymena thermophila with a mutated circular ribosomal DNA plasmid vector.

A circular plasmid containing a complete Tetrahymena thermophila rRNA gene (rDNA), with a tandem repeat of a 1.9-kilobase-pair segment encompassing the replication origin and the rRNA promoter, and a polylinker in the 3' nontranscribed spacer, was used to transform T. thermophila by microinjection. Most (20/21) stable transformants contained only recombinant linear palindromic rDNA molecules carrying rDNA sequences from both the donor plasmid and the recipient cell, as shown previously. However, in one transformant, the circular plasmid initially outreplicated the endogenous rDNA and was the major rDNA form for up to 65 generations. Stable circular replicons have not been reported previously in Tetrahymena. A single point mutation (+G) was identified in the repeated promoter of the plasmid maintained in this transformant. After recovery from the Tetrahymena transformant and recloning in Escherichia coli, the mutated circular plasmid again transformed Tetrahymena with stable maintenance of the circular rDNA plasmid. Transformants containing circular replicons were also obtained by using a similar plasmid from which the repeated promoter, but not the repeated replication origin, had been removed by BAL-31 deletion. We therefore propose that repeated rRNA promoters are deleterious in vivo in Tetrahymena, which normally lacks them. Transformants were obtained in 2-5 days compared with the 7-14 days required for transformation with unmutated rDNA plasmids by recombination. Similar results were obtained when a 550-base-pair segment containing the telomerase RNA gene of T. thermophila was inserted in the polylinker of the plasmid. We suggest that this plasmid is a useful vector system for transformation of Tetrahymena.

Animals↗
PubMed1985

Complete DNA sequence of lymphotropic papovavirus: prototype of a new species of the polyomavirus genus.

Lymphotropic papovavirus (LPV) is a new member of the polyomavirus genus. Its host range in vitro is restricted to transformed cells of B-lymphocyte origin. Here the complete 5270-bp DNA sequence of LPV is presented. The LPV early region can encode a large T and a small t antigen but no middle T antigen and the late region can encode the three structural proteins VP1, VP2, and VP3. Based on sequence conservation of shared proteins LPV is equally related to both mouse polyomavirus (Py) and simian virus 40 (SV40) and represents a new distinct species of the polyomavirus genus. Sequence comparisons of LPV, SV40, and Py point out essential conserved sequence features of the polyomavirus genus more clearly than the comparison of only SV40 and Py. The most conserved proteins are VP1 with 42% and large T antigen with 28% of the amino acids conserved among the three viruses. Although least conserved the noncoding DNA sequences of LPV show significant homologies both to SV40 and Py (origin of viral DNA replication and putative early promoter). A 63-bp tandem repeat at the late side of the replication origin possibly represents a LPV enhancer element.

Amino Acid Sequence↗
PubMed2003

Multiple functional elements comprise a Mammalian chromosomal replicator.

The structure of replication origins in metazoans is only nominally similar to that in model organisms, such as Saccharomyces cerevisiae. By contrast to the compact origins of budding yeast, in metazoans multiple elements act as replication start sites or control replication efficiency. We first reported that replication forks diverge from an origin 5' to the human c-myc gene and that a 2.4-kb core fragment of the origin displays autonomous replicating sequence activity in plasmids and replicator activity at an ectopic chromosomal site. Here we have used clonal HeLa cell lines containing mutated c-myc origin constructs integrated at the same chromosomal location to identify elements important for DNA replication. Replication activity was measured before or after integration of the wild-type or mutated origins using PCR-based nascent DNA abundance assays. We find that deletions of several segments of the c-myc origin, including the DNA unwinding element and transcription factor binding sites, substantially reduced replicator activity, whereas deletion of the c-myc promoter P1 had only a modest effect. Substitution mutagenesis indicated that the sequence of the DNA unwinding element, rather than the spacing of flanking sequences, is critical. These results identify multiple functional elements essential for c-myc replicator activity.

Animals↗
PubMed2004

Characterization of a novel ATR-dependent, Chk1-independent, intra-S-phase checkpoint that suppresses initiation of replication in Xenopus.

In most eukaryotes, replication origins fire asynchronously throughout S-phase according to a precise timing programme. When replication fork progression is inhibited, an intra-S-phase checkpoint is activated that blocks further origin firing and stabilizes existing replication forks to prevent them undergoing irreversible collapse. We show that chromatin incubated in Xenopus egg extracts displays a replication-timing programme in which firing of new replication origins during S phase depends on the continued activity of S-phase-inducing cyclin-dependent kinases. We also show that low concentrations of the DNA-polymerase inhibitor aphidicolin, which only slightly slows replication-fork progression, strongly suppress further initiation events. This intra-S-phase checkpoint can be overcome by caffeine, an inhibitor of the ATM/ATR checkpoint kinases, or by neutralizing antibodies to ATR. However, depletion or inhibition of Chk1 did not abolish the checkpoint. We could detect no significant effect on fork stability when this intra-S-phase checkpoint was inhibited. Interestingly, although caffeine could prevent the checkpoint from being activated, it could not rescue replication if added after the timing programme would normally have been executed. This suggests that special mechanisms might be necessary to reverse the effects of the intra-S-phase checkpoint once it has acted on particular origins.

Animals↗
PubMed2006

A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene.

We confirmed that the replication of the fragile-X E site (FRAXE) in human chromosomal band Xq28 occurs at six hours into the eight-hour S phase of normal human fibroblasts. In this late-replicating region, we mapped an origin of DNA replication within the promoter of FMR2. This origin is coincident with CpG islands, the trinucleotide repeat, and exon 1 of the FMR2 gene. Identification of this origin may aid in the investigation of the mechanism of trinucleotide repeat expansion and its effect on FMR2 expression. In addition, knowledge of the chromosomal locations and sequence characteristics of both early and late origins of DNA replication, such as the one described in this report, will facilitate studies of the molecular determinants of the time of activation of different origins of replication and allow us to refine our insights concerning origin inactivation in response to the DNA damage-induced intra-S checkpoint.

Cell Line↗
PubMed2006

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus.

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.

Caulobacter crescentus↗
PubMedDate not supplied

Initiation of chromosome replication: structure and function of oriC and DnaA protein in eubacteria.

Recent advances in DNA technology have made it possible to analyse the structure and function of the replication origin region of the chromosomes of various bacteria. Comparative studies have shown that 2 basic elements, the replicator and initiator, involved in initiation of chromosome replication are common to most eubacteria but with differences in the fine organization of these elements. In this article, we first review studies of the structural analysis of the origin regions of bacterial chromosomes, and then we summarize our recent work on the function of the 2 elements in Bacillus subtilis as compared to Escherichia coli, in order to show how organization of the elements is related to the differences in regulation of the initiation of replication in the 2 bacteria. Remarkable conservation of genes and their organization in the replication origin region was found in 5 bacteria representative of 3 major branches of the bacterial phylogenic tree. It was concluded that the conserved region containing the dnaA gene is the replication origin of the ancestral bacterium. Conservation of DnaA protein and its binding sequence (DnaA box) is remarkable, suggesting that they are the initiator and replicator of the chromosomes of most eubacteria. We have recently isolated an autonomously replicating sequence (ars) from B. subtilis. The essential features of ars, the presence of DnaA boxes and repeats of an AT rich 15-mer, are the same as E. coli oriC. However, 2 DnaA-box regions flanking the dnaA gene are both required for B. subtilis ars. The function of DnaA protein in vivo was studied in detail using a temperature-sensitive dnaA mutant in B. subtilis.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis↗