Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone.

We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.     

High-throughput mapping of origins of replication in human cells

Mapping origins of replication has been challenging in higher eukaryotes. We have developed a rapid, genome-wide method to map origins of replication in asynchronous human cells by combining the nascent strand abundance assay with a highly tiled microarray platform, and we validated the technique by two independent assays. We applied this method to analyse the enrichment of nascent DNA in three 50-kb regions containing known origins of replication in the MYC, lamin B2 (LMNB2) and haemoglobin beta (HBB) genes, a 200-kb region containing the rare fragile site, FRAXA, and a 1,075-kb region on chromosome 22; we detected most of the known origins and also 28 new origins. Surprisingly, the 28 new origins were small in size and located predominantly within genes. Our study also showed a strong correlation between origin replication timing and chromatin acetylation.     

Identification of a predominant replication origin in fission yeast.

We have identified five autonomously replicating sequences (ARSs) in a 100 kbp region of the Schizosaccharomyces pombe chromosome II. Analyses of replicative intermediates of the chromosome DNA by neutral/neutral two-dimensional gel electrophoresis demonstrated that at least three of these ARS loci operate as chromosomal replication origins. One of the loci,ori2004, was utilized in almost every cell cycle, while the others were used less frequently. The frequency of initiation from the respective chromosomal replication origin was found to be roughly proportional to the efficiency of autonomous replication of the corresponding ARS plasmid. Replication from ori2004 was initiated within a distinct region almost the same as that for replication of the ARS plasmid. These results showed that the ori2004 region of approximately 3 kbp contains all the cis elements essential for initiation of chromosome replication.     

Differential activity of two non-hr origins during replication of the baculovirus Autographa californica nuclear polyhedrosis virus genome

The identification of potential baculovirus origins of replication (ori) has involved the generation and characterization of defective interfering particles that contain major genomic deletions yet retain their capability to replicate by testing the replication ability of transiently transfected plasmids carrying viral sequences in infected cells. So far, there has not been any evidence to demonstrate the actual utilization of these putative origins in Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) replication. By using the method of origin mapping by competitive PCR, we have obtained quantitative data for the ori activity of the HindIII-K region and the ie-1 promoter sequence in AcMNPV. We also provide evidence for differential activity of the two ori in the context of the viral genome through the replication phase of viral infection. Comparison of the number of molecules representing the HindIII-K and ie-1 origins vis-à-vis the non-ori polH region in a size-selected nascent DNA preparation revealed that the HindIII-K ori is utilized approximately 14 times more efficiently than the ie-1 region during the late phase of infection. HindIII-K also remains the more active ori through the early and middle replication phases. Our results provide in vivo evidence in support of the view that AcMNPV replication involves multiple ori that are activated with vastly different efficiencies during the viral infection cycle.     

Cloning and sequence analysis of a plasmid replicon from Lactococcus lactis subsp. cremoris FG2

A replication region from one of the Lactococcus lactis subsp. cremoris FG2 plasmids was isolated by cloning of a 4.8-kb XbaI fragment into a replication probe vector and transformation into L. lactis LM0230. A 1.8-kb region within this fragment was sequenced and confirmed by PCR subcloning to encode a functional replicon in LM0230. The replicon consists of an open reading frame encoding a putative replication protein (Rep) of 386 amino acids and a non-coding region (ori) which features several structural motifs typical of other known replication origins, including a 22-bp iteron sequence tandemly repeated three and a half times, a 10-bp direct repeat and two sets of inverted repeats. The ori region could drive replication of its plasmid when supplied with the replication region in-trans. The lack of detectable single-stranded DNA during replication and the existence of extensive homology with other known lactococcal theta replicons strongly suggest that this region encodes a theta-replicating mechanism.     

Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences

Strain PT23 of Pseudomonas syringae pv, tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c. 61 kb (c. 74%) of pPT23B is repeated in pPT23A and only c. 17 kb (c. 21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid of P. syringae pv. syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P. syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P. syringae strains.     

Isolation and characterization of a DNA replication origin from the 1,700-kilobase-pair symbiotic megaplasmid pSym-b of Rhizobium meliloti.

A 4-kb fragment active as an autonomously replicating sequence (ARS) from the Rhizobium meliloti symbiotic megaplasmid pSym-b was isolated by selecting for sequences that allowed a normally nonreplicative pBR322 derivative to replicate in R. meliloti. The resulting Escherichia coli-R. meliloti shuttle plasmid (mini-pSym-b) containing the ARS also replicated in the closely related Agrobacterium tumefaciens, but only in strains carrying pSym-b, suggesting that a megaplasmid-encoded trans-acting factor is required. The copy number of mini-pSym-b was approximately the same as that of the resident megaplasmid, and mini-pSym-b was unstable in the absence of antibiotic selection. An 0.8-kb DNA subfragment was sufficient for replication in both R. meliloti and A. tumefaciens. The minimal ARS exhibited several sequence motifs common to other replication origins, such as an AT-rich region, three potential DnA binding sites, a potential 13-mer sequence, and several groups of short direct repeats. Hybridization experiments indicated that there may be a related ARS on the other megaplasmid, pSym-a. The pSym-b ARS was mapped near exoA, within a region nonessential for pSym-b replication. These results suggest that the R. meliloti megaplasmids share conserved replication origins and that pSym-b contains multiple replication origins. Since the mini-pSym-b shuttle vector can coexist with IncP-1 broad-host-range plasmids, it is also now possible to use two compatible plasmids for cloning and genetic manipulation in R. meliloti.     

Influence of a replication enhancer on the hierarchy of origin efficiencies within a cluster of DNA replication origins.

DNA replication origins in animal cells sometimes occur in clusters. Often one of the multiple origins within these clusters fires more frequently than the others. The reason for this hierarchy remains unknown. Similar origin clusters occur in the fission yeast, Schizosaccharomyces pombe. One such cluster is located near the ura4 gene on chromosome III and contains three origins: ars3002, ars3003, and ars3004. In their natural chromosomal context (ars3003 is about 2.5 kb upstream of ars3002 and ars3004 is adjacent to ars3002 on the downstream side) their initiation frequencies display a striking hierarchy: ars3002 > ars3003 > ars3004. Here, we describe experiments that reveal a 400 bp replication enhancer within ars3004, adjacent to ars3002. The enhancer is essential for ars3004 origin function in a plasmid, but even with the enhancer ars3004 is an inefficient origin. The enhancer is not essential for ars3002 plasmid origin activity, but dramatically stimulates this activity, converting ars3002 from an inefficient plasmid origin to a very efficient one. It also stimulates the plasmid origin activity of ars3001 and ars3003 at all tested positions and orientations on both sides of each autonomously replicating sequence (ARS) element. If ars3002 is redefined to include the enhancer, then the relative activities of the three ARS elements as single origins within separate plasmids or as origins when all three ARS elements are present in a single plasmid is the same as the chromosomal hierarchy. Thus, this replication enhancer defines the relative activities of the three origins in the ura4 origin region. Similar enhancers may affect relative activities in the origin clusters of animal cells.     

The DNA replication origins of herpes simplex virus type 1 strain Angelotti

The nucleotide sequences of the origins of DNA replication (ori) of the S- and L-component (oriS, oriL) of the herpes simplex virus type 1 (HSV-1) standard genome were determined from HSV-1 strain Angelotti (ANG). In contrast to other HSV-1 strains, the ANG oriS sequence revealed an insertion of an TA-dinucleotide in an otherwise very conserved but imperfect palindromic sequence of 47 bp. The oriL sequence of the standard ANG genome was found to be identical to that of an ANG class II defective genome which exhibits a duplication of a 144 bp palindrome. A model is presented to explain the origination of the amplified ANG oriL sequences from the parental genome with a single copy of oriL via illegitimate recombination. Alignment of the ori sequences of HSV, adeno- and papovaviruses unveiled that the HSV ori region can be subdivided into two distinct sites of homology to the DNA initiation signals of papova- and adenoviruses, suggesting that the HSV origins of replication comprise elements for DNA replication by both, cellular and virus-encoded DNA polymerases.     

Characterization of a phage-plasmid hybrid (phasyl) with two independent origins of replication isolated from Escherichia coli.

The phage-plasmid hybrid phasyl can replicate as a phage in the presence of a filamentous phage of Escherichia coli (M13, fl, fd). The extragenic region of phasyl shows homology with the plus and the minus origins of filamentous phages. Insertion of a Cmr fragment into the plus origin or of a Kmr fragment into the minus origin resulted in a reduced transduction frequency, while insertion into other parts of the extragenic region did not. This suggests that phagelike replication of phasyl is mediated by an origin that coincides with the two homologous elements in the extragenic region. Autonomous replication of phasyl occurs from a second origin (oriA) that is located between positions 297 and 636. This fragment mediates replication if the Arp protein is supplied in trans. Arp is the only phage-encoded protein and is essential for plasmidlike replication. No sequence homology to other known origins was found. Phasyl derivatives with either one of the two origins inactivated can be rescued via the alternative replication mode, suggesting that the two replication pathways are independent.     

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.     

Localization of a DNA replication origin and termination zone on chromosome III of Saccharomyces cerevisiae.

Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome III. Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins. A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin). Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.     

Restriction maps and homologies of the three plasmids of Agrobacterium rhizogenes strain A4

Agrobacterium rhizogenes strain A4 is a virulent agropine-type strain possessing three plasmids: plasmid a (pArA4a, 180 kb) is not necessary for plant transformation, plasmid b (250 kb) is the root-inducing plasmid (pRiA4), and plasmid c (pArA4c) is a cointegrate of pArA4a and pRiA4. The total plasmid DNA (pArA4) of strain A4 was cloned in the cosmid pHSG262 and the library obtained was used to establish BamHI maps of the three plasmids. The plasmids a and Ri have an apparently identical region and a partly homologous region, and are different in the remaining regions including their origins of replication. Another agropine-type A. rhizogenes strain, HRI, bears only one plasmid, which is the Ri plasmid (pRiHRI). pRiHRI and pRiA4 present the same restriction maps for a great part, but are different in a region of 48 kb; however, this region of pRiHRI is found unmodified in pArA4a and may have a role in the virulence of the bacteria. The comparison between the restriction maps of the plasmids of strain A4 leads us to propose that the recombination event leading to pArA4c formation occurs within the identical regions of pArA4a and pRiA4. In addition, the comparison with the already established map of pRiHRI suggests that strain HRI could have been derived from a recombination event between the two homologous regions of pArA4c with subsequent loss of the smaller plasmid.     

Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

DNA replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict and often tissue-specific temporal program. However, the genetic program that controls activation of replication origins has still not been fully elucidated in mammalian cells. Previously, we measured replication timing at the sequence level along human chromosomes 11q and 21q. In the present study, we sought to obtain a greater understanding of the relationship between replication timing programs and human chromosomes by analysis of the timing of replication of a single human chromosome 11 that had been transferred into the Chinese hamster ovary (CHO) cell line by chromosome engineering. Timing of replication was compared for three 11q chromosomal regions in the transformed CHO cell line (CHO(h11)) and the original human fibroblast cell line, namely, the R/G-band boundary at 11q13.5/q14.1, the centromere and the distal telomere. We found that the pattern of replication timing in and around the R/G band boundary at 11q13.5/q14.1 was similar in CHO(h11) cells and fibroblasts. The 11q centromeric region, which replicates late in human fibroblasts, replicated in the second half of S phase in CHO(h11) cells. By contrast, however, the telomeric region at 11q25, which is late replicating in fibroblasts (and in several other human cell lines), replicated in the first half of S phase or in very early S phase in CHO(h11) cells. Our observations suggest that the replication timing programs of the R/G-band boundary and the centromeric region of human chromosome 11q are maintained in CHO(h11) cells, whereas that for the telomeric region is altered. The replication timing program of telomeric regions on human chromosomes might be regulated by specific mechanisms that differ from those for other chromosomal regions.     

Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain

We have used two complementary two-dimensional gel electrophoretic methods to localize replication inititation sites and to determine replication fork direction in the amplified 240 kb dihydrofolate reductase domain of the methotrexate-resistant CHO cell line CHOC 400. Surprisingly, our analysis indicates that replication begins at many sites in several restriction fragments distributed throughout a previously defined 28 kb initiation locus, including a fragment containing a matrix attachment region. Initiation sites were not detected in regions lying upstream or downstream of this locus. Our results suggest that initiation reactions in mammalian chromosomal origins may be more complex than in the origins of simple microorganisms.