Bidirectional replication from an internal ori site of the linear N15 plasmid prophage

The prophage of coliphage N15 is not integrated into the chromosome but exists as a linear plasmid molecule with covalently closed hairpin ends (telomeres). Upon infection the injected phage DNA circularizes via its cohesive ends. Then, a phage-encoded enzyme, protelomerase, cuts the circle and forms the hairpin telomeres. N15 protelomerase acts as a telomere-resolving enzyme during prophage DNA replication. We characterized the N15 replicon and found that replication of circular N15 miniplasmids requires only the repA gene, which encodes a multidomain protein homologous to replication proteins of bacterial plasmids replicated by a theta-mechanism. Replication of a linear N15 miniplasmid also requires the protelomerase gene and telomere regions. N15 prophage replication is initiated at an internal ori site located within repA and proceeds bidirectionally. Electron microscopy data suggest that after duplication of the left telomere, protelomerase cuts this site generating Y-shaped molecules. Full replication of the molecule and subsequent resolution of the right telomere then results in two linear plasmid molecules. N15 prophage replication thus appears to follow a mechanism that is distinct from that employed by eukaryotic replicons with this type of telomere and suggests the possibility of evolutionarily independent appearances of prokaryotic and eukaryotic replicons with covalently closed telomeres.     

Remodeling of the human papillomavirus type 11 replication origin into discrete nucleoprotein particles and looped structures by the E2 protein

The human papillomavirus (HPV) DNA replication origin (ori) shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundreds of base pairs. The HPV type 11 ori spans 103 bp and contains three palindromic E2 binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 ori binding protein. These sites are separated by 64 and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 DNA ori bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound, indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies on the dependence of looping on salt, ATP, and DTT using full-length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N-terminal domain and factors that may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.     

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.     

Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921

pSA1.1 is a 9.1-kb multicopy plasmid originally isolated from Streptomyces cyaneus (formerly S. azureus) ATCC 14921. This plasmid accumulates single-stranded DNA in S. lividans and is therefore considered to replicate by a rolling-circle replication. In the present work, the rep gene encoding the replication initiator protein and the replication origin ori of pSA1.1 were determined. The rep and ori are located on separate regions. The Rep protein of pSA1.1 belongs to superfamily I which includes A proteins of phages. Nucleotide sequence of the surrounding putative nicking site of pSA1.1 shows good agreement with those of the pC194 group plasmids and phages. The direction of replication was also determined.     

Kaposi's sarcoma-associated herpesvirus ori-Lyt-dependent DNA replication: involvement of host cellular factors

Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.     

The ori sequences of the mitochondrial genome of a wild-type yeast strain: number,location, orientation and structure

We have investigated the number, the location, the orientation and the structure of the seven ori sequences present in the mitochondrial genome of a wild-type strain, A, of Saccharomyces cerevisiae. These homologous sequences are formed by three G + C-rich clusters. A, B and C, and by four A + T-rich stretches. Two of the latter, p and s, are located between clusters A and B; one, l between clusters B and C; and one r, either immediately follows cluster C (in ori 3–7), or is separated from it by an additional A + T-rich stretch, r', (in ori 1 and ori 2). The most remarkable differences among ori sequences concern the presence of two additional G + C-rich clusters,β and γ, which are inserted in sequence l of ori 4 and 6 and in the middle of sequence r of ori 4,6 and 7, respectively. Neglecting clusters /gb and γ and stretch r', the length of on sequences is 280 ± 1 bp, and that of the l stretch 200 ± 1 bp. Hairpin structures can be formed by the whole A-B region, by clusters β and γ, and (in ori 2–6) by a short AT sequence, lp, immediately preceding cluster /gb. An overall tertiary folding of ori sequences can be obtained. Some structural features of ori sequences are shared by the origins of replication of the heavy strands of the mitochondrial genomes of mammalian cells.     

The Two Cis-Acting Sites,parS1 and oriC1, Contribute to the Longitudinal Organisation of Vibrio cholerae Chromosome I

The segregation of bacterial chromosomes follows a precise choreography of spatial organisation. It is initiated by the bipolar migration of the sister copies of the replication origin (ori). Most bacterial chromosomes contain a partition system (Par) with parS sites in close proximity to ori that contribute to the active mobilisation of the ori region towards the old pole. This is thought to result in a longitudinal chromosomal arrangement within the cell. In this study, we followed the duplication frequency and the cellular position of 19 Vibrio cholerae genome loci as a function of cell length. The genome of V. cholerae is divided between two chromosomes, chromosome I and II, which both contain a Par system. The ori region of chromosome I (ori_I) is tethered to the old pole, whereas the ori region of chromosome II is found at midcell. Nevertheless, we found that both chromosomes adopted a longitudinal organisation. Chromosome I extended over the entire cell while chromosome II extended over the younger cell half. We further demonstrate that displacing parS sites away from the ori_I region rotates the bulk of chromosome I. The only exception was the region where replication terminates, which still localised to the septum. However, the longitudinal arrangement of chromosome I persisted in Par mutants and, as was reported earlier, the ori region still localised towards the old pole. Finally, we show that the Par-independent longitudinal organisation and ori_I polarity were perturbed by the introduction of a second origin. Taken together, these results suggest that the Par system is the major contributor to the longitudinal organisation of chromosome I but that the replication program also influences the arrangement of bacterial chromosomes.

Author summary

Proper chromosome organisation within the cell is crucial for cellular proliferation. However, the mechanisms driving bacterial chromosome segregation are still strongly debated, partly due to their redundancy. Two patterns of chromosomal organisation can be distinguished in bacteria: a transversal chromosomal arrangement, such as in E. coli, where the origin of replication (ori) is positioned at midcell and flanked by the two halves of the chromosome (replichores), and a longitudinal arrangement, such as in C. crescentus, where ori is recruited to the pole and the replichores extend side by side along the long axis of the cell. Here, we present the first detailed characterization of the arrangement of the genetic material in a multipartite genome bacterium. To this end, we visualised the position of 19 loci scattered along the two V. cholerae chromosomes. We demonstrate that the two chromosomes, which both harbour a Par system, are longitudinally organised. However, the smaller one only extended over the younger cell half. In addition, we found that disruption of the Par system of chromosome I released its origin from the pole but preserved its longitudinal arrangement. Finally, we show that the addition of an ectopic ori perturbed this arrangement, suggesting that the replication program contributes to chromosomal organisation.     

Straight core structure of DNA replication origins in the mammalian AMPD2 locus

Identification of the nucleotide consensus sequence in mammalian replication origins is a difficult and controversial problem. The hypothesis that local DNA topology could be involved in recognition by replication proteins is an exciting possibility. Secondary DNA structures, including intrinsically bent DNA, can be easily detected, and they may indicate a specific pattern in or near mammalian replication origins. This work presents the entire mapping of the intrinsically bent DNA sites (IBDSs), using in silico analysis and a circular permutation assay, of the DNA replication origins oriGNAI3, oriC, oriB, and oriA in the mammalian amplified AMPD2 gene domain. The results show that each origin presents an IBDS that flanks the straight core of these DNA replication sites. In addition, the in silico prediction of the nucleosome positioning reveals a strong indication that the center of an IBDS is localized in a nucleosome-free region (NFR). The structure of each of these curved sites is presented together with their helical parameters and topology. Together, the data that we present here indicate that the oriGNAI3 origin where preferential firing to the replication initiation events in the amplified AMPD2 domain occurs is the only origin that presents a straight, narrow region that is flanked on both sides by two intrinsically bent DNA sites within a short distance (～300 bp); however, all of the origins present at least one IBDS, which is localized in the NFR region. These results indicate that structural features could be implicated in the mammalian DNA replication origin and support the possibility of detecting and characterizing these segments.     

Replication of bacteriophage lambda DNA. Examination of variants containing double origins and observation of a bias in directionality

Bacteriophage λ variants have been constructed that possess two λ ori sites. Replicative intermediates resulting from infection with these phages have been investigated. We find that initiation of replication from the ori site on an EcoRI fragment (containing all the DNA sequences from within the red gene to the middle of gene O) cloned in the inverted orientation is predominantly bidirectional but occurs at a decreased frequency. Double initiations were observed at low frequency. However, a second cloned ori fragment (carrying two large deletions and a small insertion) cloned in the normal orientation demonstrated insignificant levels of replication from the cloned site unless the normal ori had already initiated.A bias in directionality of λ replication has been observed. Molecules that replicate unidirectionally propagate to the right more often than to the left. If the cloned ori-containing EcoRI fragment is inserted with reversed polarity, then the bias is towards the left. Bidirectional λ replicative intermediates also appear to show a similar bias but this is superimposed on a large, apparently random, effect that results in asymmetric growing-point propagation.     

Excision sequences in the mitochondrial genome of yeast

We report an analysis of the sequences used in the excision of the mitochondrial genomes of 22 spontaneous and ten ethidium bromide (EtBr)-induced Saccharomyces cerevisiae petite mutants. In all cases, excision sequences were found to be perfect direct repeats, often flanked on one or both sides by regions of patchy homology. Sequences used in the excision of the genomes of spontaneous petites were always located in the AT spacers and GC clusters of intergenic regions of the genome; the GC clusters corresponded to ori and ori^s sequences, namely to canonical and surrogate origins of DNA replication, respectively. In the case of the ethidium bromide-induced petites, excision sequences were found not only in intergenic sequences, but also in the introns and exons of mitochondrial genes.