Replication of R6K γ origin in vitro: discrete start sites for DNA synthesis dependent on π and its copy-up variants 1 1Edited by G. Smith

Chen, Dongzhao; Feng, Jinhong; Krüger, Ricardo Henrique; Urh, Marjeta; Inman, Ross B.; Filutowicz, Marcin

doi:10.1006/jmbi.1998.2055

Cited by 22 publications

(18 citation statements)

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“…Accordingly, we used a pFW25 vector that contains the R6K ␥ origin (␥ ori) of replication (34). The ␥ ori is a unidirectional origin, which can function only in the presence of the protein encoded by the pir gene (45). In E. coli ECF005 and ECF006, the protein is expressed from a chromosomal pir gene, which is under the control of an arabinose promoter (see "Experimental Procedures").…”

Section: Frequency Of Recombination Of Triplet Repeatsmentioning

confidence: 99%

“…The reason why the relative orientations of the CTG⅐CAG repeats have such a dramatic effect on their recombinogenicity is uncertain but may be due to a residual amount of replication from the R6K origin. However, literature exists (45,84) that argues against this possibility. Another possibility, albeit remote, is that the secondary structure of the triplet repeat tract is somehow different in orientation I as compared with orientation II and that these differences may present a barrier to the recombination machinery.…”

Section: Instability Of the Ctg⅐cag Tracts In The Recombinationmentioning

confidence: 99%

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Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

Pluciennik

Iyer

Напиерала

et al. 2002

Journal of Biological Chemistry

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Homologous recombination was shown to enable the expansion of CTG⅐CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG⅐CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG⅐CAG) n repeats apparently recombine with an ϳ60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG⅐CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG⅐CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.Micro-and minisatellite instability has been associated with human genetic diseases (1-4). Approximately 14 hereditary neurological diseases are caused by the genetic instabilities of triplet repeat sequences (TRS) 1 in or near relevant genes (reviewed in Ref. 1). Long tracts of repeating CTG⅐CAG sequences are responsible for myotonic dystrophy and several other diseases. Normal individuals have 5-37 repeats in the myotonic dystrophy protein kinase gene, whereas the mutations (expansions) can be in the range of 50 -3000 repeats. Thus, an explosive allele length change during intergenerational transmission can occur in this autosomal dominant disease, thus causing an increase in the severity of the disease and a decrease of the age at onset (anticipation).The mechanisms of genetic instabilities have been widely investigated in the past 6 years (1). DNA replication (5-8) and repair including methyl-directed mismatch repair (9 -11), nucleotide excision repair (12), DNA polymerase III exonucleolytic proofreading (13), and double-strand break repair (14) have been implicated.Repetitive sequences promote homologous recombination in prokaryotic as well as eukaryotic systems, presumably by virtue of forming unusual DNA secondary structures (15)(16)(17)(18)(19)(20)(21)(22). In fact, homologous recombination has been implicated in the instability of repetitive sequences (23-26). Similar findings have also been made with CTG⅐CAG repeats using a two-plasmid system in Escherichia coli (27,28). Multiple fold expansions, deletions, and the exchange of point mutations between tracts were found in this system; these events were dependent on the presence of TRS tracts on both plasmids, CTG⅐CAG repeat lengths longer than 30, and a functional recA gene.Previously (29), it was proposed that CTG⅐CAG tracts might function as recombination hot spots in the bovine genome. More recently, Young et al. (30) surveyed the genome of Saccharomyces cerevisiae and suggested that these repeats cou...

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Section: Frequency Of Recombination Of Triplet Repeatsmentioning

confidence: 99%

Section: Instability Of the Ctg⅐cag Tracts In The Recombinationmentioning

confidence: 99%

Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

Pluciennik

Iyer

Напиерала

et al. 2002

Journal of Biological Chemistry

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“…1). Possible mechanisms for iteronbased inhibition (handcuffing [44]) and non-iteron-based inhibition (repressing primer synthesis [this paper]) have been proposed (3,11). To determine which of these potential mechanisms of inhibition would be triggered first, we conducted quantitative binding assays.…”

Section: Emsa Reveals Binding Of Derivatives Of Various Molecular Weimentioning

confidence: 99%

“…Next we examined whether copy-up variants of 35.0 can bind to the non-iteron site. We undertook comparative binding studies to determine if a correlation exists between the non-iteron binding abilities (or lack thereof) of copy-up variants and their known elevated replication activities in vitro (3). An inverse correlation would mean that the higher activity of copy-up might be explained, at least in part, by a reduced ability to repress primer synthesis in the AϩT-rich region.…”

Section: Emsa Reveals Binding Of Derivatives Of Various Molecular Weimentioning

confidence: 99%

“…1). One of the predictions of such a model would be that copy-up mutants, which stimulate in vitro replication more than the wild-type counterpart (3,28), might do so as a consequence of the decreased or altered binding to the noniteron site. This hypothesis seemed attractive in view of our recent findings suggesting that the structures of wild-type 35.0 dimers and dimers 35.0 containing copy-up substitutions are most likely different (44).…”

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Dimers of π Protein Bind the A+T-Rich Region of the R6K γ Origin near the Leading-Strand Synthesis Start Sites: Regulatory Implications

Krüger

Filutowicz

2000

J Bacteriol

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The replication of ␥ origin, a minimal replicon derived from plasmid R6K, is controlled by the Rep protein . At low intracellular concentrations, activates the ␥ origin, while it inhibits replication at elevated concentrations. Additionally, acts as a transcription factor (auto)repressing its own synthesis. These varied regulatory functions depend on binding to reiterated DNA sequences bearing a TGAGNG motif. However, also binds to a "non-iteron" site (i.e., not TGAGNG) that resides in the A؉T-rich region adjacent to the iterons. This positioning places the non-iteron site near the start sites for leading-strand synthesis that also occur in the A؉T-rich region of ␥ origin. We have hypothesized that origin activation (at low levels) would require the binding of monomers to iterons, while the binding of dimers to the non-iteron site (at high levels) would be required to inhibit priming. Although monomers as well as dimers can bind to an iteron, we demonstrate that only dimers bind to the non-iteron site. Two additional pieces of data support the hypothesis of negative replication control by binding to the non-iteron site. First, binds to the non-iteron site about eight times less well than it binds to a single iteron. Second, hyperactive variants of protein (called copy-up) either do not bind to the non-iteron site or bind to it less well than wild-type . We propose a replication control mechanism whereby would directly inhibit primer formation.Recognition of the replication origin (ori) by an initiator protein is the central event regulating DNA synthesis in diverse biological systems. The ␥ ori of the antibiotic resistance plasmid R6K belongs to a large group of replicons that regulate replication through initiator proteins (Rep proteins) which bind to reiterated DNA sequences (iterons) (8). Although R6K contains multiple oris, the ␥ ori is a faithful model for studying the regulation of replication because it retains the copy control and stability loci of the parental plasmid (22,49).Regulated replication of a basic ␥ ori replicon depends upon protein encoded by the plasmid's pir gene. The pir gene encodes two in-frame polypeptides (55)

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PlasmidDNAReplication

Tolmasky

Actis

Crosa

1999

Encyclopedia of Bioprocess Technology

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Replication of R6K γ origin in vitro: discrete start sites for DNA synthesis dependent on π and its copy-up variants 1 1Edited by G. Smith

Cited by 22 publications

References 51 publications

Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

Dimers of π Protein Bind the A+T-Rich Region of the R6K γ Origin near the Leading-Strand Synthesis Start Sites: Regulatory Implications

PlasmidDNAReplication

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