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PubMed2004

Genomic specification and epigenetic regulation of eukaryotic DNA replication origins.

Identification of DNA replication origins (ORIs) at a genome-wide level in eukaryotes has proved to be difficult due to the high degree of degeneracy of their sequences. Recent structural and functional approaches, however, have circumvented this limitation and have provided reliable predictions of their genomic distribution in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and they have also significantly increased the number of characterized ORIs in animals. This article reviews recent evidence on how ORIs are specified and maintained in these systems and on their regulation and sensitivity to epigenetic signals. It also discusses the possible additional involvement of ORIs in processes other than DNA replication.

AT Rich Sequence↗
PubMed1994

DNA helical instability facilitates initiation at the SV40 replication origin.

Previous analysis of mutations in bacterial and yeast replication origins has identified a genetic component, termed a DNA unwinding element (DUE), whose intrinsic helical instability is essential for origin function. For the SV40 replication origin, we show here that the early palindrome (EP) and A + T-rich (AT) domains both exhibit helical instability, despite their dissimilar A + T compositions. To test the possible contribution of helical instability to SV40 origin function, the relationship between helical stability of mutant origin sequences and their known origin activity in vitro and in vivo was examined. Origin activity correlates inversely with the helical stability of mutations within the EP domain but not the AT domain or the T-antigen binding domain. The quantitative correlation holds for four different measures of origin activity determined in vitro and in vivo. An even better-correlated collection of mutations was found in a specific portion of the EP domain. This specific EP subdomain coincides with the sequence known to be strand-separated after T-antigen binds the origin in vitro and with the origin of bidirectional replication in vivo. Our analysis of origin mutations indicates that the helical instability of the specific EP subdomain is required to facilitate T-antigen-induced melting and the initiation of DNA replication. The sensitivity of the required EP subdomain to mutations that stabilize the DNA helix defines the DUE of the SV40 replication origin.

Adenine↗
PubMed1993

The effect on chromosome stability of deleting replication origins.

The observed spacing between chromosomal DNA replication origins in Saccharomyces cerevisiae is at least four times shorter than should be necessary to ensure complete replication of chromosomal DNA during the S phase. To test whether all replication origins are required for normal chromosome stability, the loss rates of derivatives of chromosome III from which one or more origins had been deleted were measured. In the case of a 61-kb circular derivative of the chromosome that has two highly active origins and one origin that initiates only 10 to 20% of the time, deletion of either highly active origin increased its rate of loss two- to fourfold. Deletion of both highly active origins caused the ring chromosome to be lost in approximately 20% of cell divisions. This very high rate of loss demonstrates that there are no efficient cryptic origins on the ring chromosome that are capable of ensuring its replication in the absence of the origins that are normally used. Deletion of the same two origins from the full-length chromosome III, which contains more than six replication origins, had no effect on its rate of loss. These results suggest that the increase in the rate of loss of the small circular chromosome from which a single highly active origin was deleted was caused by the failure of the remaining highly active origin to initiate replication in a small fraction (approximately 0.003) of cell cycles.

Chromosome Mapping↗
PubMed1994

Two steps in the assembly of complexes at yeast replication origins in vivo.

The integration of chromosomal DNA replication into the eukaryotic cell cycle might involve temporal regulation of interactions between cellular factors and replication origins. We show here that yeast replication origins exist in two chromatin states during the cell cycle. In the postreplicative state, genomic footprints closely resemble those produced in vitro by the purified ORC and ABF1 proteins, indicating that the binding of these proteins to replication origins is not sufficient to drive the initiation of DNA replication. The prereplicative state is characterized by an additional region of protection overlapping the ORC footprint. This prereplicative complex appears near the end of mitosis and persists through G1. After entry into S phase, origins return to the postreplicative state. Similarities in temporal regulation of the prereplicative state and the Xenopus licensing factor suggest that mechanisms limiting DNA replication to once per cell cycle may be conserved among eukaryotes.

Base Sequence↗
PubMed1995

Effects of the pSC101 partition (par) locus on in vivo DNA supercoiling near the plasmid replication origin.

Previous work has shown that deletion of the partition (par) locus of plasmid pSC101 results in decreased overall superhelical density of plasmid DNA and concommitant inability of the plasmid to be stably inherited in populations of dividing cells. We report here that the biological effects of par correlate specifically with its ability to generate supercoils in vivo near the origin of pSC101 DNA replication. Using OsO4 reactivity of nucleotides adjoining 20 bp (G-C) tracts introduced into pSC101 DNA to measure local DNA supercoiling, we found that the wild type par locus generates supercoiling near the plasmid's replication origin adequate to convert a (G-C) tract in the region to Z form DNA. A 4 bp deletion that decreases par function, but produces no change in the overall superhelicity of pSC101 DNA as determined by chloroquine/agarose gel analysis, nevertheless reduced (G-C) tract supercoiling sufficiently to eliminate OsO4 reactivity. Mutation of the bacterial topA gene, which results in stabilized inheritance of par-deleted plasmids, restored supercoiling of (G-C) tracts in these plasmids and increased OsO4 reactivity in par+ replicons. Removal of par to a site more distant from the origin decreased supercoiling in a (G-C) tract adjacent to the origin and diminished par function. Collectively, these findings indicate that par activity is dependent on its ability to produce supercoiling at the replication origin rather than on the overall superhelical density of the plasmid DNA.

Bacterial Proteins↗
PubMed1997

Analysis of the tobacco chloroplast DNA replication origin (oriB) downstream of the 23 S rRNA gene.

We have mapped the origin of DNA replication (oriB) downstream of the 23 S rRNA gene in each copy of the inverted repeat (IR) of tobacco chloroplast DNA between positions 130,502 and 131,924 (IR(A)) by a combination of approaches. In vivo chloroplast DNA replication intermediates were examined by two-dimensional agarose gel electrophoresis. Extended arc patterns suggestive of replication intermediates containing extended single-stranded regions were observed with the 4.29 kb SspI fragment and an overlapping EcoRI fragment from one end of the inverted repeat, while only simple Y patterns were observed with a 3.92 kb BamHI-KpnI fragment internal to the SspI fragment. Other restriction fragments of tobacco chloroplast DNA besides those at the oriA region also generated only simple Y patterns in two-dimensional agarose gels. Several chloroplast DNA clones from this region were tested for their ability to support in vitro DNA replication using a partially purified chloroplast protein fraction. Templates with a deletion of 154 bp from the SspI to the BamHI sites near the end of the inverted repeat resulted in a considerable loss of in vitro DNA replication activity. These results support the presence of a replication origin at the end of the inverted repeat. The 5' end of nascent DNA from the replication displacement loop was identified at position 130,697 for IR(A) (111,832 for IR(B)) by primer extension. A single major product insensitive to alkali and RNase treatment was observed and mapped to the base of a stem-loop structure which contains one of two neighboring BamHI sites near the end of each inverted repeat. This provides the first precise determination of the start site of DNA synthesis from oriB. Adjacent DNA fragments containing the stem-loop structure and the 5' region exhibit sequence-specific gel mobility shift activity when incubated with the replication protein fraction, suggesting the presence of multiple binding sites.

Base Sequence↗
PubMed1996

Transcriptional terminators of RNA polymerase II are associated with yeast replication origins.

The compact organization of the Saccharomyces cerevisiae genome necessitates that non-coding regulatory sequences reside in close proximity to one another. Here we show there is an intimate association between transcription terminators and DNA replication origins. Four replication origins were analyzed in a reporter gene assay that detects sequences that direct 3' end formation of mRNA transcripts. All four replication origins function as orientation-independent transcription terminators in this system, producing truncated polyadenylated mRNAs. Despite this close association, the cis-acting elements that confer replication origin function are genetically separable from those required for transcription termination. Several models are explored in an attempt to address how and why the signals specifying transcription termination and replication initiation overlap.

Base Sequence↗
PubMed2002

The origin recognition complex marks a replication origin in the human TOP1 gene promoter.

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the human TOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation.

Base Sequence↗
PubMed1985

Conformational changes in a replication origin induced by an initiator protein.

The replication initiator protein of the plasmid R6K binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins alpha, beta, and gamma. Binding of the initiator to the direct repeats induced a marked bending of the region of gamma replication origin. Binding of the initiator also promoted unwinding of the origin DNA by at least two turns. Distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. At the appropriate DNA and protein concentrations the initiator enhanced topoisomerase-induced catenation of the origin containing supercoiled DNA but not of DNA lacking the origin sequence. Thus, the initiator protein caused significant changes in the secondary and tertiary structures of the replication origin.

Bacterial Proteins↗
PubMed2004

Eucaryotic replication origin binding proteins.

Initiation of eukaryotic DNA replication is a tightly controlled process. Replication initiates at multiple specific sites (replication origins) that have been licensed for replication, following the cell cycle-dependent, multi-step assembly of specific factors. Thus, replication origins occur in two chromatin states: a replication-competent pre-replicative (pre-RC) state, when a number of replication proteins assemble on the origin in a stepwise fashion, and a replication-incompetent post-replicative (post-RC) state, in which the origin (or elements of it) is bound only by the origin recognition complex (ORC) (or subunits of it). This review summarizes the origin binding proteins that have been have been identified to date.

14-3-3 Proteins↗
PubMed1990

Alpha and beta replication origins of plasmid R6K show similar distortions of the DNA helix in vivo.

Plasmid R6K contains inverted repeats of an approximately 100-base-pair sequence separated by 5.5 kilobases. These long inverted repeats (LIRs) occur within the alpha and beta origins of replication and are essential for origin function. In this study, primer-extension analysis of DNA modified in vivo by dimethyl sulfate or KMnO4 revealed that both alpha and beta LIRs acquire similar structural distortions of the DNA helix in a functional R6K replicon. These distortions were not seen in plasmids containing isolated LIR sequences. In the functional replicon, the dimethyl sulfate and KMnO4 hyperreactive sites appear on complementary strands and are located to one side of an internal palindromic sequence within the LIRs. This asymmetry coincides with the primary direction of DNA replication from alpha and beta origins in vivo. We also observed two intermediate structures when certain R6K cis- or trans-acting elements are missing. Sequences near the alpha origin are required for generation of the dimethyl sulfate hyperreactive sites, whereas sequences near the beta origin are responsible for the appearance of KMnO4 hyperreactive sites. We suggest that these structures represent a hierarchy that leads to a "locked" preinitiation complex, which functions to synchronize and determine the direction of replication from the alpha and beta replication origins in vivo.

Base Sequence↗
PubMed1998

DNA loop anchorage region colocalizes with the replication origin located downstream to the human gene encoding lamin B2.

The recently developed procedure of topoisomerase II-mediated DNA loop excision has been used to analyze the topological organization of a human genome fragment containing the gene encoding lamin B2 and the ppv1 gene. A 3.5 kb long DNA loop anchorage/topoisomerase II cleavage region was found within the area under study. This region includes the end of the lamin B2 coding unit and an intergenic region where an origin of DNA replication was previously found. These observations further corroborate the hypothesis that DNA replication origins are located at or close to DNA loop anchorage regions.

Binding Sites↗
PubMed1993

Minimal replication origin of the 200-kilobase Halobacterium plasmid pNRC100.

We have identified the replication origin of pNRC100, a 200-kb plasmid of Halobacterium halobium, by assaying for replication ability of miniplasmids containing cloned fragments of pNRC100 and the mevinolin resistance selectable marker of Haloferax volcanii. First, we showed the replication ability of plasmid pNGHCMEV1, which contains the 19-kb HindIII-C fragment of pNRC100, by recovery of plasmid DNA from mevinolin-resistant transformants of H. halobium. The minimal replication origin of approximately 3.9 kb was defined by subcloning successively smaller regions of pNGHCMEV1 and assaying for plasmid replication in either H. halobium or H. volcanii. The same replication origin was also recovered after transformation of H. volcanii with a library of partial Sau3AI fragments of pNRC100. The nucleotide sequence of the minimal replication origin was determined and found to contain a long open reading frame, named repH, transcribed away from a highly A+T-rich region. The transcription start site was identified by primer extension analysis to be 17 to 18 nucleotides 5' to a putative repH start codon. The predicted product of the repH gene, an acidic protein with a molecular weight of 113,442, showed 24 to 27% identity with predicted gene products of H. volcanii plasmid pHV2 and H. halobium plasmid p phi HL, suggesting that each is involved in plasmid replication. One pNRC100 minireplicon, pNG11 delta 12, was analyzed by linker scanning mutagenesis, which showed the requirement of repH for replication. Restoration of the repH reading frame of one replication-defective pNG11 delta 12 derivative by introduction of a second small insertion resulted in reversion to replication proficiency. The replication ability of pNG11delta12 was lost when the entire A+T-rich region, about 550 bp long, was deleted but not when small insertions or deletions were introduced into this region. The presence of only 52 bp of the A+T-rich segment was sufficient to permit replication. The pNG11delta12 minireplicon was lost at high frequency from cells grown without mevinolin selection, suggesting that the plasmid partitioning locus of pNRC100 is absent in the minimal replication origin region. We discuss the possible roles of the repH gene and the A+T-rich region in replication of pNRC100.

Amino Acid Sequence↗
PubMed1996

Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum.

We analyzed the replication of two unlinked actin genes, ardB and ardC , which are abundantly transcribed in the naturally synchronous plasmodium of the slime mold Physarum polycephalum. Detection and size measurements of single-stranded nascent replication intermediates (RIs) demonstrate that these two genes are concomitantly replicated at the onset of the 3-h S phase and tightly linked to replication origins. Appearance of RIs on neutral-neutral two-dimensional gels at specific time points in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, site-specific, bidirectional origin of replication is localized within the promoter region of the gene. We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon. Finally, taking advantage of a restriction fragment length polymorphism, we studied allelic replicons and demonstrate similar localizations and a simultaneous firing of allelic replication origins. Computer search revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the yeast autonomously replicating sequence consensus sequence in these Physarum origin regions. Our results with the ardB and ardC actin genes support the model of early replicating origins located within the promoter regions of abundantly transcribed genes in P. polycephalum.

Actins↗
PubMed1996

Composite patterns in neutral/neutral two-dimensional gels demonstrate inefficient replication origin usage.

The neutral/neutral two-dimensional (2-D) gel replicon mapping technique has been used to great advantage to localize and characterize origins of replication. Interestingly, many yeast origins display a composite pattern consisting of both a bubble arc and a single-fork arc. Moreover, in every instance in which neutral/neutral 2-D gels have been used to analyze origins in higher eukaryotic cells, two or more adjacent fragments display these composite patterns. We believe that composite patterns signal inefficient origin usage in yeast cells because the replicators in question are not active in every cell cycle and in higher eukaryotic replicons because initiation sites are chosen from among many potential sites lying within a zone. However, others have suggested that the single-fork arcs in these composite gel patterns arise from nicking activity that converts replication bubbles to branched structures that comigrate with bona fide single forks. Here, we have used three different replicon mapping strategies to show that broken simian virus 40 replication bubbles trace unique arcs that are clearly distinguishable from classic, intact single forks. Thus, it is likely that composite 2-D gel patterns represent origins that are inefficiently utilized.

Animals↗
PubMed1977

Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy.

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.

DNA Replication↗
PubMed1974

More precise mapping of the replication origin in Escherichia coli K-12.

The origin of replication in Escherichia coli K-12 was mapped by determining the rate of marker replication during a synchronous round of replication. Four isogenic strains were made lysogenic for lambdaind(-) and for phage Mu-1, with Mu-1 integrated into a different chromosomal location in each strain. Cultures were starved for amino acids to allow completion of chromosome replication cycles and then starved for thymine in the presence of amino acids, and a synchronous cycle of replication was initiated by the addition of thymine. Samples were exposed to radioactive thymidine at intervals, deoxyribonucleic acid was extracted, and the rate of marker replication was determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization to filters containing Mu-1, lambda, and E. coli deoxyribonucleic acid. The results confirm that the origin of replication is near ilv. The travel times of the replication forks, calculated from the data obtained for cultures with doubling times of approximately 40 and 61 min, are 40 and 52 min, respectively.

Amino Acids↗