Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta. The ratio of approximately 0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta'). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta, ori-beta', and ori-gamma), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.     

Transformation abrogates an early G1-phase arrest point required for specification of the Chinese hamster DHFR replication origin

The origin decision point (ODP) was originally identified as a distinct point during G1-phase when Chinese hamster ovary (CHO) cell nuclei experience a transition that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. Passage of cells through the ODP requires a mitogen-independent protein kinase that is activated prior to restriction point control. Here we show that inhibition of an early G1-phase protein kinase pathway by the addition of 2-aminopurine (2-AP) prior to the ODP arrests CHO cells in G1-phase. Transformation with simian virus 40 (SV40) abrogated this arrest point, resulting in the entry of cultured cells into S-phase in the presence of 2-AP and a disruption of the normal pattern of initiation sites at the DHFR locus. Cells treated with 2-AP after the ODP initiated replication specifically within the DHFR origin locus. Transient exposure of transformed cells to 2-AP during the ODP transition also disrupted origin choice, whereas non-transformed cells arrested in G1-phase and then passed through a delayed ODP after removal of 2-AP from the medium. We conclude that mammalian cells have many potential sites at which they can initiate replication. Normally, events occurring during the early G1-phase ODP transition determine which of these sites will be the preferred initiation site. However, if chromatin is exposed to S-phase-promoting factors prior to this transition, mammalian cells, like Xenopus and Drosophila embryos, can initiate replication without origin specification.     

Distal sequences, but not ori-beta/OBR-1, are essential for initiation of DNA replication in the Chinese hamster DHFR origin

In the Chinese hamster dihydrofolate reductase replication initiation zone, the ori-beta locus is preferred over other start sites. To test the hypothesis that ori-beta contains a genetic replicator, we restored a deletion in the 3' end of the DHFR gene with a cosmid that provides the missing sequence and simultaneously knocks out the downstream ori-beta locus. Replication initiates normally in ori-beta knockout cell lines, and the DHFR domain is still synthesized in early S phase. However, initiation is completely suppressed in the starting deletion variant lacking the 3' end of the gene. We conclude that ori-beta does not contain an essential replicator, but that distant sequence elements have profound effects on origin activity in this locus.     

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.     

oriGNAI3: a narrow zone of preferential replication initiation in mammalian cells identified by 2D gel and competitive PCR replicon mapping techniques

The nature of mammalian origins of DNA replication remains controversial and this is primarily because two-dimensional gel replicon mapping techniques have identified broad zones of replication initiation whereas several other techniques, such as quantitative PCR, have disclosed more discrete sites of initiation at the same chromosomal loci. In this report we analyze the replication of an amplified genomic region encompassing the 3'-end of the GNAI3 gene, the entire GNAT2 gene and the intergenic region between them in exponentially growing Chinese hamster fibroblasts. These cells express GNAI3 but not GNAT2 . The replication pattern was first analyzed by two-dimensional neutral-alkaline gel electrophoresis. Surprisingly, the results revealed a small preferential zone of replication initiation, of at most 1.7 kb, located in a limited part of the GNAI3 - GNAT2 intergenic region. Mapping of this initiation zone was then confirmed by quantitative PCR. The agreement between the two techniques exploited here strengthens the hypothesis that preferred sites of replication initiation do exist in mammalian genomes.     

Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places

DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.     

Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.     

Genetic dissection of a mammalian replicator in the human beta-globin locus

The timing and localization of DNA replication initiation in mammalian cells are heritable traits, but it is not known whether initiation requires specific DNA sequences. A site-specific recombination strategy was used to show that DNA sequences previously identified as replication initiation sites could initiate replication when transferred to new chromosomal locations. An 8-kilobase DNA sequence encompassing the origin of DNA replication in the human beta-globin locus initiated replication in the simian genome. Specific deletions within the globin origin did not initiate replication in these chromosomal sites. These data suggest that initiation of DNA replication in mammalian cells requires specific sequence information and extend the replicon hypothesis to higher eukaryotes.     

A computer model for the analysis of DNA replication intermediates by two-dimensional agarose gel electrophoresis

We present a computer model to predict the patterns expected for the replication intermediates (RIs) of DNA fragments analyzed by neutral/neutral two-dimensional (2D) agarose gel electrophoresis. The model relies on the mode of replication (uni- or bi-directional), the electrophoretic mobility of linear DNA fragments and the retardation caused by the three-dimensional shape of non-linear molecules. The utility of this model is demonstrated with two examples: replication analysis of the plasmids pBR322 and pHH5.8 in Escherichia coli after digestions with EcoRI and HindIII, respectively.     

Developing a model of DNA replication to be used for Monte Carlo calculations that predict the sizes and shapes of molecules resulting from DNA double-strand breaks induced by X irradiation during DNA synthesis

A Monte Carlo computer program was written to introduce double-strand breaks (DSBs) randomly into cellular DNA that is configured according to different models of DNA replication. Then, from a review of the literature using DNA fiber autoradiography and other studies relating to rates of replication of DNA that is organized in approximately 3-Mbp regions or bands, a particular model for DNA replication was developed. Using this model, Monte Carlo calculations were made to predict the types and sizes of molecules that would result from introducing DSBs into DNA when synchronous cells are irradiated in the middle of S phase. Then results of the Monte Carlo calculations were compared with migration profiles obtained by pulsed-field gel electrophoresis (PFGE) for molecular size distributions of linear DNA molecules. For these comparisons, CHO cells irradiated in S phase also were pulse-labeled at the time of irradiation with [3H]dThd for 15 min to compare the migration patterns of 3H-labeled replicating DNA with those of the mass of S-phase DNA, measured by imaging with a CCD camera. For the Monte Carlo calculations, we assumed from the reports in the literature that molecules containing replication bubbles with and without forks would be trapped in the PFGE plug. We also assumed that those molecules that are < or = 8 Mbp, both linear and with replication forks, would be released into the lane. However, approximately 75% of the 3H-labeled DNA that is released from the plug migrated much more slowly than linear molecules, which we attributed to the slow migration of 3H-labeled molecules having replication forks not attached to bubbles. The percentages of both mass of S-phase DNA and 3H-labeled replicating DNA released from the plug, as determined by PFGE, were compared with comparable values determined from Monte Carlo calculations. A DNA replication model that provides good agreement between the PFGE results and Monte Carlo calculations is described. Furthermore, Monte Carlo methodology is presented that can be used for comparing data obtained with PFGE with results of Monte Carlo calculations that are based on different models of DNA replication and different assumptions for the migration of various types of replicating molecules.     

Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.     

In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): a remnant of bacterial rolling circle and conjugative plasmids?

According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes.     

Characterization of excised DNA intermediates associated with V(D)J recombination at the T-cell receptor delta locus

Lymphocyte development requires the assembly of antigen receptor genes through the specialized process of V(D)J recombination. This process is initiated by cleavage at the junction between coding segments (V, D, and J) and the recombination signal sequences that border these segments, resulting in generation of double-strand break intermediates. We have used a two-dimensional gel system to characterize broken molecules arising from V(D)J recombination at the T-cell receptor (TCR) delta locus and have identified linear species excised by Ddelta1-Ddelta2 and V-Ddelta2 rearrangement in thymus DNA. Relatively few (approximately 10) V-Ddelta2-excised linear species were detected in DNA from fetal thymocytes. The sizes of these species corresponded to the estimated distances between Ddelta2 and the V gene segments utilized by gammadelta T cells and indicated that both Ddelta2-proximal and -distal V gene segments are targeted for V-Ddelta2 rearrangement. Similar-sized species were observed in DNA from thymocytes of scid mice in which T-cell development is arrested prior to TCR expression. Since previous studies suggest that the TCR alpha/delta locus encodes more than 100 V gene segments, our results indicate that a few select V gene segments are predominantly targeted for rearrangement to Ddelta2, and this primarily accounts for the restricted Vdelta gene repertoire of gammadelta T cells.     

In search of circular permuted variants of Escherichia coli dihydrofolate reductase

A circularized form of a Cys-free mutant of Escherichia coli dihydrofolate reductase (DHFR) was used to search for a proteolytic site that gave new N- and C-termini on circularized DHFR with enzyme activity. Of the six site-specific proteolytic enzymes tested, three proteases, Achromobacter protease I (lysine-specific endopeptidase), asparaginylendopeptidase, and Staphylococcus aureus V8 protease, cleaved a single site of the circularized DHFR to form circular permuted variants. Twenty-four possible sites for cleavage were found formation of eight circular permuted variants was suggested by results of N-terminal sequence analysis of the linearized proteins isolated by gel filtration in the presence of 5 M guanidine hydrochloride. Mapping of the predicted cleavage sites on the DHFR molecule suggested that they were not all at a specific loop and, therefore, there are many possible circular permuted variants.     

Semi-conservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template

We describe the preparation of nuclear extracts from yeast cells synchronised in S-phase that support the aphidicolin-sensitive, semi-conservative replication of primer-free, supercoiled plasmid in vitro. This is monitored by one and two-dimensional gel electrophoresis of replication intermediates that have incorporated [alpha-32P]dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by substitution of dTTP with the heavy derivative BrdUTP, which results in a shift in density corresponding to complete second strand synthesis. We demonstrate dependence on DNA pol delta and the pol alpha/primase complex, and are able to detect putative Okazaki fragments under ATP-limiting conditions. In contrast to the semi-conservative replication of supercoiled plasmid, linear or open-circular templates incorporate labelled nucleotides through repair synthesis that produces no significant density shift on CsCl gradients. Consistent with a true replication reaction we find that semi-conservative replication of plasmid DNA is stimulated in S-phase relative to G1-phase nuclear extracts, and is independent of the recombination-promoting factor Rad52p. Using this novel system we demonstrate that semi-conservative replication, but not polymerase activity per se, requires the activity of the DNA helicase encoded by DNA2.