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1.
Inverted repeats as genetic elements for promoting DNA inverted duplication: implications in gene amplification 总被引:5,自引:0,他引:5
Inverted repeats are important genetic elements for genome instability. In the current study we have investigated the role of inverted repeats in a DNA rearrangement reaction using a linear DNA substrate. We show that linear DNA substrates with terminal inverted repeats can efficiently transform Escherichia coli. The transformation products contain circular inverted dimers in which the DNA sequences between terminal inverted repeats are duplicated. In contrast to the recombination/rearrangement product of circular DNA substrates, which is exclusively one particular form of the inverted dimer, the rearrangement products of the linear DNA substrate consist of two isomeric forms of the inverted dimer. Escherichia coli mutants defective in RecBCD exhibit much reduced transformation efficiency, suggesting a role for RecBCD in the protection rather than destruction of these linear DNA substrates. These results suggest a model in which inverted repeats near the ends of a double-strand break can be processed by a helicase/exonuclease to form hairpin caps. Processing of hairpin capped DNA intermediates can then yield inverted duplications. Linear DNA substrates containing terminal inverted repeats can also be converted into inverted dimers in COS cells, suggesting conservation of this type of genome instability from bacteria to mammalian cells. 相似文献
2.
A plasmid containing inverted repeats is constructed in Bacillus subtilis. Insertion of DNA fragments into the plasmid inverted repeats results either in the precise excision of the insert or in its duplication in the opposite inverted repeat. These rearrangements are due to the presence of inverted repeats only. Two recombination events are possibly responsible for these phenomena. During the first step of the recombination two plasmid monomers form a dimer molecule. During the second step the intramolecular recombination between the direct repeats in the dimer structure leads to the formation of two rearranged plasmid monomers devoid of insertion or containing two DNA inserts. Proposed dimeric intermediate is unstable in B. subtilis. However, it is isolated from Escherichia coli recA, cells. Plasmids containing the inverted repeats can serve as a model to study plasmid recombination in B. subtilis cells. 相似文献
3.
Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA from embryos of the sea urchin Strongylocentrotus intermedius, extruded DNA enriched for replication origins was obtained. The average length of the fragments of the DNA of this fraction was estimated to be about 800 base pairs. The origin-enriched nascent DNA strands were assayed for the presence of inverted repeats. The results show that the origin-enriched DNA is also enriched in inverted repeats. The bulk of palindromes in the total nuclear DNA was estimated to be 200 base pairs in length and from the origin-enriched DNA-150 base pairs. 相似文献
4.
Intrastrand annealing leads to the formation of a large DNA palindrome and determines the boundaries of genomic amplification in human cancer
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Tanaka H Cao Y Bergstrom DA Kooperberg C Tapscott SJ Yao MC 《Molecular and cellular biology》2007,27(6):1993-2002
Amplification of large chromosomal regions (gene amplification) is a common somatic alteration in human cancer cells and often is associated with advanced disease. A critical event initiating gene amplification is a DNA double-strand break (DSB), which is immediately followed by the formation of a large DNA palindrome. Large DNA palindromes are frequent and nonrandomly distributed in the genomes of cancer cells and facilitate a further increase in copy number. Although the importance of the formation of large DNA palindromes as a very early event in gene amplification is widely recognized, it is not known how a DSB is resolved to form a large DNA palindrome and whether any local DNA structure determines the location of large DNA palindromes. We show here that intrastrand annealing following a DNA double-strand break leads to the formation of large DNA palindromes and that DNA inverted repeats in the genome determine the efficiency of this event. Furthermore, in human Colo320DM cancer cells, a DNA inverted repeat in the genome marks the border between amplified and nonamplified DNA. Therefore, an early step of gene amplification is a regulated process that is facilitated by DNA inverted repeats in the genome. 相似文献
5.
Spontaneous deletion mutations often occur at short direct repeats that flank inverted repeat sequences. Inverted repeats may initiate genetic rearrangements by formation of hairpin secondary structures that block DNA polymerases or are processed by structure-specific endonucleases. We have investigated the ability of inverted repeat sequences to stimulate deletion of flanking direct repeats in Escherichia coli. Propensity for cruciform extrusion in duplex DNA correlated with stimulation of flanking deletion, which was partially sbcD dependent. We propose two mechanisms for palindrome-stimulated deletion, SbcCD dependent and SbcCD independent. The SbcCD-dependent mechanism is initiated by SbcCD cleavage of cruciforms in duplex DNA followed by RecA-independent single-strand annealing at the flanking direct repeats, generating a deletion. Analysis of deletion endpoints is consistent with this model. We propose that the SbcCD-independent pathway involves replication slipped mispairing, evoked from stalling at hairpin structures formed on the single-stranded lagging-strand template. The skew of SbcCD-independent deletion endpoints with respect to the direction of replication supports this hypothesis. Surprisingly, even in the absence of palindromes, SbcD affected the location of deletion endpoints, suggesting that SbcCD-mediated strand processing may also accompany deletion unassociated with secondary structures. 相似文献
6.
Mechanism of autonomous control of the Escherichia coli F plasmid: purification and characterization of the repE gene product. 总被引:5,自引:1,他引:4
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E protein, the 29 Kd repE gene product, is essential for the replication of the Escherichia coli F plasmid. The repE gene has been cloned and expressed in an inducible ATG-fusion vector, and the protein product has been purified to homogeneity. The purified E protein is present as a dimer in solution and binds specifically to both the 19-bp direct repeats (incB) found within the mini-F origin of replication ori2 and the repE operator DNA. Examination of the amino acid sequence of E protein revealed a consensus sequence for DNA binding. 相似文献
7.
Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1. 相似文献
8.
The data concerning the biological functions and properties of short specific polynucleotide sequences (so called insertion sequences--IS) are reviewed. IS elements integrated in a genome can lead to strongly polar mutations in Escherichia coli, its bacteriophages and plasmids, while some IS (IS2) being integrated in inverted orientation turn on the gene activity. Several copies of the IS elements are present in the E. coli chromosome. A characteristic feature of IS is their ability to recA-independent migration along the bacterial chromosome. Possible mechanisms of IS integration are discussed. IS elements play the key role in the majority of recA-independent recombinational events: F-prime and partially Hfr-formation, plasmid recombination and dissociation, some cases of deletion formation etc. IS elements participate in recombination in the form of direct or inverted repeats. Direct repeats probably determine the processes of dissociation of the complete multicomponent R-factors and other plasmids. Inverted repeats (some of them are palindromes) are responsible for the migration of several drug-resistance determinants called transposons. Possible mechanisms of IS-dependent and probably IS-controlled recombination are discussed. 相似文献
9.
Origin of adenovirus DNA replication. Role of the nuclear factor I binding site in vivo 总被引:12,自引:0,他引:12
R T Hay 《Journal of molecular biology》1985,186(1):129-136
An assay is described that detects in vivo a single round of initiation and DNA synthesis directed by a linear molecule containing an exposed single copy of an adenovirus (Ad) origin of replication. This and a previously described assay, which measures multiple rounds of DNA replication, were used to identify DNA sequences within the Ad2 and Ad4 origins of replication that are important for ori function. Linear DNA molecules containing sequences from the Ad2 or Ad4 genome termini were cotransfected with homologous and heterologous helper virus, and net amounts of DNA synthesis were compared. Linear molecules containing the Ad4 inverted terminal repeats were replicated 20-fold better in the presence of the homologous helper, whereas both Ad2 and Ad4 inverted terminal repeats were utilized efficiently by Ad4. DNA sequence analysis of the Ad2 ori and the corresponding region in Ad4 indicated that, although there are only ten variant base-pairs, eight are located within the Ad2 DNA sequence recognized by the cellular protein nuclear factor I. This protein is required to achieve the maximal rate of Ad2 DNA replication in vitro, and these differences therefore identify DNA sequences that are crucial to Ad2 ori function. The Ad4 ITR does not contain a functional nuclear factor I binding site, and deletion analysis has demonstrated that this region of the Ad4 genome is not required for ori function. In contrast to Ad2, the DNA sequences required for the initiation of Ad4 DNA replication were shown to reside entirely within the terminal 18 base-pairs of the Ad4 inverted terminal repeat. 相似文献
10.
《Cell cycle (Georgetown, Tex.)》2013,12(2):114-116
Pol I is the most abundant polymerase in E. coli and plays an important role in short patch repair. In accord with this role in the cell, the purified polymerase exhibits low processivity and high fidelity in vitro. Pol I is also the polymerase responsible for leader strand synthesis during ColE1 plasmid replication. In a previous publication, we described the generation of a highly error-prone DNA polymerase I. Expression of this mutant Pol I results in errors during the replication of a ColE1 plasmid. The distribution and spectrum of mutations in the ColE1 plasmid sequence downstream the ori indicates that Pol I is capable of more processive replication in vivo than previously accepted. Here, we review evidence suggesting that Pol I may be recruited into a replisome-like holoenzyme and speculate that processive DNA replication by Pol I in the cell may play a role in recombination-dependent DNA replication. 相似文献
11.
Pol I is the most abundant polymerase in E. coli and plays an important role in short patch repair. In accord with this role in the cell, the purified polymerase exhibits low processivity and high fidelity in vitro. Pol I is also the polymerase responsible for leader strand synthesis during ColE1 plasmid replication. In a previous publication, we described the generation of a highly error-prone DNA polymerase I. Expression of this mutant Pol I results in errors during the replication of a ColE1 plasmid. The distribution and spectrum of mutations in the ColE1 plasmid sequence downstream the ori indicates that Pol I is capable of more processive replication in vivo than previously accepted. Here, we review evidence suggesting that Pol I may be recruited into a replisome-like holoenzyme and speculate that processive DNA replication by Pol I may play a role in recombination-dependent DNA replication in the cell. 相似文献
12.
Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model
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Cheung AK 《Journal of virology》2004,78(17):9016-9029
Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome. 相似文献
13.
Identification of the origin of replication of the eukaryote Dictyostelium discoideum nuclear plasmid Ddp2 总被引:1,自引:0,他引:1
Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryote Dictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication in D. discoideum, provided that the Rep gene, which acts in trans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp "TGTCATGACA" palindromes separated by a poly(T.A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication. 相似文献
14.
15.
D K Chattoraj 《Cell》1980,19(1):143-149
Biparental lambda phage DNA dimers formed by the Rec recombination system of E. coli were isolated in the absence of DNA replication and phage maturation. The RecA but not the RecB gene is required for dimer formation. Dimers are primarily circular but can also be branched circular or linear. In circular dimers the crossover points are distributed uniformly along the chromosome, even in the presence of the RecB-dependent Chi recombinational hotspots. Thus in the absence of DNA synthesis and maturation, the Rec system can act reciprocally both in the presence and absence of the RecB gene; this lack of RecB participation accounts for the observed lack of Chi activity. 相似文献
16.
Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus 总被引:2,自引:0,他引:2
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Cheung AK 《Journal of virology》2004,78(8):4268-4277
Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle "melting-pot" model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a "melted" configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a "gene correction" or "terminal repeat correction" mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules. 相似文献
17.
On the Deletion of Inverted Repeated DNA in Escherichia Coli: Effects of Length, Thermal Stability, and Cruciform Formation in Vivo 总被引:9,自引:2,他引:7
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We have studied the deletion of inverted repeats cloned into the EcoRI site within the CAT gene of plasmid pBR325. A cloned inverted repeat constitutes a palindrome that includes both EcoRI sites flanking the insert. In addition, the two EcoRI sites represent direct repeats flanking a region of palindromic symmetry. A current model for deletion between direct repeats involves the formation of DNA secondary structure which may stabilize the misalignment between the direct repeats during DNA replication. Our results are consistent with this model. We have analyzed deletion frequencies for several series of inverted repeats, ranging from 42 to 106 bp, that were designed to form cruciforms at low temperatures and at low superhelical densities. We demonstrate that length, thermal stability of base pairing in the hairpin stem, and ease of cruciform formation affect the frequency of deletion. In general, longer palindromes are less stable than shorter ones. The deletion frequency may be dependent on the thermal stability of base pairing involving approximately 16-20 bp from the base of the hairpin stem. The formation of cruciforms in vivo leads to a significant increase in the deletion frequency. A kinetic model is presented to describe the relationship between the physical-chemical properties of DNA structure and the deletion of inverted repeats in living cells. 相似文献
18.
The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends. 相似文献
19.
Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryoteDictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication inD. discoideum, provided that the Rep gene, which acts intrans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp “TGTCATGACA” palindromes separated by a poly(T · A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication. 相似文献
20.
David J. Pinder Catherine E. Blake Janet C. Lindsey & David R. F. Leach 《Molecular microbiology》1998,28(4):719-727
We have isolated and sequenced a set of deletions stimulated by DNA palindromes in Escherichia coli . All of the deletions are asymmetric with respect to the parental sequence and have occurred at short direct repeats. This is consistent with deletion by strand slippage during DNA replication. The orientation of the asymmetry in such deletion products is diagnostic of the direction of the strand slippage event. It is therefore also diagnostic of its occurrence on the leading or lagging strand of the replication fork when the direction of replication is known. In all cases in which the orientation of the asymmetry could be determined with respect to DNA replication, the products were consistent with a preference for deletion on the lagging strand of the fork. The data include replication slippage in three situations: on the chromosome of E . coli , in bacteriophage λ and in high-copy-number pUC-based plasmids. 相似文献