Identification and functional analysis of a human homologue of the monkey replication origin ors8

We previously isolated from African green monkey (CV-1) cells a replication origin, ors8, that is active at the onset of S-phase. Here, its homologous sequence (hors8, accession number: DQ230978) was amplified from human cells, using the monkey-ors8-specific primers. Sequence alignment between the monkey and the human fragment revealed a 92% identity. Nascent DNA abundance analysis, involving quantification by real-time PCR, indicated that hors8 is an active replication origin, as the abundance of nascent DNA from a genomic region containing it was 97-fold higher relative to a non-origin region in the same locus. Furthermore, the data showed that the hors8 fragment is capable of supporting the episomal replication of its plasmid, when cloned into pBlueScript (pBS), as assayed by the DpnI resistance assay after transfection of HeLa cells. A quantitative chromatin immunoprecipitation (ChIP) assay, using antibodies against Ku, Orc2, and Cdc6, showed that these DNA replication initiator proteins were associated in vivo with the human ors8 (hors8). Finally, nascent DNA abundance experiments from human cells synchronized at different phases of the cell cycle revealed that hors8 is a late-firing origin of DNA replication, having the highest activity 8 h after release from late G(1).     

Structural properties of the beta origin of replication of plasmid R6K

The beta origin of replication of plasmid R6K, one of three active R6K origins of replication, requires most or all of a 1962-base pair (bp) sequence for activity. The nucleotide sequence of a portion of this functional beta origin was determined in an earlier study (Stalker, D., Kolter, R., and Helinski, D. (1982) J. Mol. Biol. 161, 33-43). In this work, the sequence of the remaining portion of this 1964-bp segment was obtained. In addition to its activity as an origin of replication, this sequence also contains sufficient information for autonomous replication in Escherichia coli. A 277-bp region containing seven 22-bp direct repeats is present at one end of the beta origin segment (Stalker, D., Kolter, R., and Helinski, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1150-1154) while the other end contains a 140-bp sequence that includes a relaxation complex site. The 277-bp direct repeat region is required for activity of the beta origin. The start of the beta origin of replication as mapped by electron microscopy (Crosa, J. (1980) J. Biol. Chem. 255, 11075-11077) lies approximately 1000 bp away from the 277-bp region. The pi structural gene, which makes up most of the sequence between the direct repeats and the beta origin, is required in cis for beta origin activity. The pi protein also is required for beta origin activity but can be provided in trans. The nucleotide sequence just beyond the pi structural gene and within or near the start of beta origin of replication contains an open reading frame for a 151-amino acid protein. Deletions ranging from 94 bp to 1590 bp were obtained within the 1964-bp beta origin region. In every case, the deletion results in loss of origin activity even when the deleted sequence plus adjacent regions are provided in trans. These observations suggest a requirement for a specific secondary structure over an extensive region for beta origin activity.     

The nucleotide sequence of the replication origin beta of the plasmid R6K

We h ave identified by molecular cloning a region of 283 base pairs of the HindIII 2 fragment of R6K which corresponds to the region of the replication origin beta. This 283 base-pair DNA fragment, when present contiguously with the structural gene for the replication initiation protein of R6K, encoded in the HindIII 9-15 and part of HindIII 2 restriction fragments, will support the replication of a plasmid chimera containing the pBR322 replicon in a pol Ats host at the nonpermissive temperature. The nucleotide sequence of the region of replication origin beta has been determined. The nucleotide sequence has some homology with the ori gamma region of R6K; it has a 15-base-pair homology with the replication origin of Escherichia coli.     

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IneW plasmid R388 and comparison with the related IncN plasmid R46

Summary We cloned and sequenced a 402 by DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 by of oriT was sufficient for efficient mobilization. The first 86 by of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 by perfect inverted repeat. Deletion of the first 95 by reduced the frequency of transfer by a hundred-fold. The sequence between by 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 by segment (from by 176–236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between by 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, whch coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.     

Directed evolution and identification of control regions of ColE1 plasmid replication origins using only nucleotide deletions

Genes can be mutated by altering DNA content (base changes) or DNA length (insertions or deletions). Most in vitro directed evolution processes utilize nucleotide content changes to produce DNA libraries. We tested whether gain of function mutations could be identified using a mutagenic process that produced only nucleotide deletions. Short nucleotide stretches were deleted in a plasmid encoding lacZ, and screened for increased beta-galactosidase activity. Several mutations were found in the origin of replication that quantitatively and qualitatively altered plasmid behavior in vivo. Some mutations allowed co-residence of ColE1 plasmids in Escherichia coli, and implicate hairpin structures II and III of the ColE1 RNA primer as determinants of plasmid compatibility. Thus, useful and unexpected mutations can be found from libraries containing only deletions.     

Small deletion variants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity

The emergence of multiply antibiotic-resistant microorganisms in the environment has become a serious public health threat. To address this, our lab has devised a methodology in which antimicrobial agents are transferred into unwanted cells using the process of bacterial conjugation. In the work described here, we pursued proteins that cause plasmid over-replication as potential antimicrobial agents. Our focus was on the pir-encoded pi protein of plasmid R6K that possesses both positive and negative functions in controlling gamma origin-based replication. We observed that three of four pir mutations examined, including two in-frame deletions, severely impaired negative plasmid-replication control. The resulting over-replication phenotype was particularly strong when a pir mutant was placed in cis to gamma origin. In conjugative mating experiments with several representatives of the family Enterobacteriaceae, the plasmids expressed postconjugational antimicrobial activity. The potential utility of a conjugation-based antimicrobial approach is discussed. Additionally, we describe the replication inhibitory function of a novel and useful Rep protein variant, pi*M36A;M38A, which binds iteron DNA exclusively as dimers.     

高温链霉菌质粒pTSC2的测序分析和滚环复制方式的鉴定

从猪粪堆肥中分离到一株编号为X3-3的可以在50℃高温生长的链霉菌菌株,该菌株含有一个约7kb的环型质粒pTSC2。【目的】克隆、测序和分析pTSC2,以及鉴定质粒的复制方式。【方法】利用分段克隆和引物延伸获得pTSC2的全序列,利用多序列比对寻找复制元件rep、dso和sso,利用中性转移和Southern杂交检测复制中间体。【结果】克隆和测序获得了全长为7516bp的pTSC2序列,预测编码8种蛋白,其中4种蛋白与链霉菌滚环复制的质粒pIJ101中负责复制和接合转移的蛋白非常相似。pTSC2的复制元件rep、dso和sso也与pIJ101的相似。克隆、转化变铅青链霉菌ZX7以及高温链霉菌2C证明了rep和dso为复制所必需元件。Southern杂交检测到pTSC2复制过程中积累了大量的单链DNA。【结论】高温链霉菌质粒pTSC2以滚环方式进行复制。这是首次在高温链霉菌中克隆和测序质粒,以及鉴定其复制方式。     

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein.

Replication of the gamma origin of Escherichia coli plasmid R6K requires pi protein, encoded by the R6K pir gene, and many host factors, including DnaA protein. Pi has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory function of pi is counteracted by integration host factor and a specific sequence of the origin called the enhancer. This 106-bp DNA segment contains a binding site for DnaA protein (DnaA box 1). In this study, we mutated this site to determine if it was required for the enhancer's function. Using gamma origin derivative plasmids with the DnaA box 1 altered or deleted, we show that this site is necessary to protect the origin against levels of wild-type pi protein that would otherwise inhibit replication. To show that the base substitutions in DnaA box 1 weakened the binding of DnaA, we developed a new application of the agarose gel retardation assay. This quick and easy assay has broad applicability, as shown in binding studies with DNA fragments carrying a different segment of the R6K origin, the chromosomal origin (oriC), or the pUC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays.     

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro

Summary The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.Abbreviations bp base pairs - Cb^r, Km^r, Sm^r resistance to carbenicillin, kanamycin, streptomycin, respectively - DR direct repeat     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Activation in vivo of the minimal replication origin beta of plasmid R6K requires a small target sequence essential for DNA looping.

The plasmid R6K contains three distinct origins of replication: alpha, beta, and gamma. The gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins. R6K therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome. We have discovered that plasmids containing the origins alpha and gamma required the Escherichia coli DnaA initiator protein in addition to the R6K-encoded initiator protein, Pi, and other host replisomal proteins for their maintenance in vivo. Plasmids initiating replication from origin beta required only the Pi initiator protein and other host replisomal proteins. We have exploited the differential requirement for the DnaA protein by origins gamma and beta to selectively study and localize the minimal origin beta sequences by deletion analysis as one test of a looping model of origin activation. A 64-bp region spanning the extreme -COOH terminal coding sequence of the Pi protein was found to be essential for replication in vivo in the absence of DnaA protein, consistent with the approximate physical location of the beta origin. Replication emanating from origin beta could be abolished in vivo by deletion of the 9-bp target site for Pi protein-mediated DNA looping between the gamma origin/enhancer and the distant beta origin. Electron microscopy of nascent replication intermediates generated in vivo directly confirmed our genetic localization of the beta origin. Our results strongly suggest that activation of the beta origin by a distant replication enhancer element requires a small target sequence essential for initiator protein-mediated DNA looping.     

Activity of the replication terminus of plasmid R6K in hybrid replicons in Escherichia coli.

Hybrids between the antibiotic resistance plasmid R6K and RSF2124, a derivative of plasmid ColEl were constructed in vetro. These hybrids exhibit the replication properties of both parents in Escherichia coli, including the use of either the R6K or the ColEl origin of replication during logarithmic phase growth. Incompatibility properties of both parental plasmids also are expressed by the hybrid plasmids. Analysis of replicative intermediates showed that the asymmetric terminus of R6K was functional in the hybrids. In the absence of protein synthesis where replication of the hybrid plasmid is initiated only from the ColE1 origin, the R6K terminus either prevents or severely impedes the progress of the replication fork. The activity of the R6K terminus region is expressed independent of the direction of DNA replication and in the absence of the R6K replication origin.     

Molecular cloning of replication and incompatibility regions from the R-plasmid R6K.

The construction of a physical map of R6K DNA on the basis of specific cleavage of R6K DNA by HindIII and EcoRI restriction endonucleases allowed us to determine the location of the R6K replication and drug resistance regions. Molecular cloning techniques were used to dissect the replication and incompatibility functions of R6K. This R-plasmid possesses two origins of replication, α and β, separated by a stretch of 3900 nucleotides. A region close to ori α. controls the copy number of the composite replicon. Inverted duplications which are 100 to 200 nucleotides long are found at the positions of ori α and ori β, respectively. A 1400-nucleotide long sequence within the region bounded by the inverted duplications and separate from the origins and the control region is involved in the R6K self-replication and replication under conditions of polymerase I deprivation. This region also contains some of the incompatibility genes of R6K. The sequentially asymmetrically bidirectional mode of R6K replication is due to the existence of a replication termination site. This terminator is located outside the sequences bounded by the inverted duplications and is not essential for plasmid DNA replication.     

Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid

Summary We provide evidence that a mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1, stabilizes the ParA^- and ParB^- miniplasmid of R1 pKN1562, without increasing its copy number. The system, which we have called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two protiens whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system determines in cis a basal but detectable stability.     

Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli

The replication period of Escherichia coli cells grown in rich medium lasts longer than one generation. Initiation thus occurs in the 'mother-' or 'grandmother generation'. Sister origins in such cells were found to be colocalized for an entire generation or more, whereas sister origins in slow-growing cells were colocalized for about 0.1-0.2 generations. The role of origin inactivation (sequestration) by the SeqA protein in origin colocalization was studied by comparing sequestration-deficient mutants with wild-type cells. Cells with mutant, non-sequesterable origins showed wild-type colocalization of sister origins. In contrast, cells unable to sequester new origins due to loss of SeqA, showed aberrant localization of origins indicating a lack of organization of new origins. In these cells, aberrant replisome organization was also found. These results suggest that correct organization of sister origins and sister replisomes is dependent on the binding of SeqA protein to newly formed DNA at the replication forks, but independent of origin sequestration. In agreement, in vitro experiments indicate that SeqA is capable of pairing newly replicated DNA molecules.