Identification of novel initiation sites for human DNA replication around ARSH1, a previously characterized yeast replicator

Replication of mammalian chromosomes depends on the activation of a large number of origins of DNA replication distributed along the chromosomes. We have focused our attention on a human DNA region, named ARSH1, localized to chromosome 2, that had been previously shown to act as an episomal origin in the yeast Saccharomyces cerevisiae. In the present study we have used a nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 5 kb region encompassing ARSH1. This analysis applied to a 1-1.4 kb nascent DNA strand fraction isolated from normal skin fibroblasts revealed the presence of two major initiations sites surrounding the ARSH1 region. With an equivalent DNA fraction obtained from HeLa cells, in addition to these sites, a broad initiation profile was observed which included the ARSH1 region. This DNA region however was not sufficient to support episomal replication of an ARSH1-containing plasmid transfected into HeLa cells.     

Physical mapping of an origin of bidirectional replication at the centre of the Borrelia burgdorferi linear chromosome

The Borrelia burgdorferi chromosome is linear, with telomeres characterized by terminal inverted repeats and covalently closed single-stranded hairpin loops. The replication mechanism of these unusual molecules is unknown. Previous analyses of bacterial chromosomes for which the complete sequence has been determined, including that of B. burgdorferi, revealed an abrupt switch in polarity of CG skew at known or putative origins of replication. We used nascent DNA strand analysis to physically map the B. burgdorferi origin to within a 2 kb region at the centre of the linear chromosome, and to show that replication proceeds bidirectionally from this origin. The results are consistent with replication models in which termination occurs at the telomeres after bidirectional, symmetrical elongation from the central origin. Sequences typical of origins of other bacterial chromosomes were not found at the origin of this spirochete. The most likely location of the replication origin of the linear chromosome is the 240 bp sequence between dnaA and dnaN where the switch in CG skew occurs.     

Major DNA replication initiation sites in the c-myc locus in human cells

DNA replication initiation sites and initiation frequencies over 12. 5 kb of the human c-myc locus, including 4.6 kb of new 5' sequence, were determined based on short nascent DNA abundance measured by competitive polymerase chain reaction using 21 primer sets. In previous measurements, no comparative quantitation of nascent strand abundance was performed, and distinction of major from minor initiation sites was not feasible. Two major initiation sites were identified in this study. One predominant site has been located at approximately 0.5 kb upstream of exon 1 of the c-myc gene, and a second new major site is located in exon 2. The site in exon 2 has not been previously identified. In addition, there are other sites that may act as less frequently used initiation sites, some of which may correspond to sites in previous reports. Furthermore, a comparison of the abundance of DNA replication intermediates over this same region of the c-myc locus between HeLa and normal skin fibroblast (NSF) cells indicated that the relative distribution was very similar, but that nascent strand abundance in HeLa cells was approximately twice that in NSF relative to the abundance at the lamin B2 origin. This increased activity at initiation sites in the c-myc locus may mainly be influenced by regulators at higher levels in transformed cells like HeLa.     

DNA replication initiates non-randomly at multiple sites near the c-myc gene in HeLa cells.

The origin of replication of the c-myc gene in HeLa cells was previously identified at low resolution within 3.5 kb 5' to the P1 promoter, based on replication fork polarity and the location of DNA nascent strands. To define the initiation events in the c-myc origin at higher resolution the template bias of nascent DNAs in a 12 kb c-myc domain has been analyzed by hybridization to strand specific probes. Strong switches in the asymmetry of nascent strand template preference confirm that replication initiates non-randomly at multiple sites within 2.4 kb 5' to the c-myc P1 promoter, and at other sites over a region of 12 kb or more. The strongest template biases occur in the 2.4 kb region 5' of the P1 promoter, shown earlier to contain sequences which allow the autonomous semiconservative replication of c-myc plasmids. An asymmetric pyrimidine heptanucleotide consensus sequence has been identified which occurs 12 times in the c-myc origin zone, and whose polarity exactly correlates with the polarity of nascent strand synthesis.     

Structure and linkage of the D2 dopamine receptor and neural cell adhesion molecule genes on human chromosome 11q23.

The gene encoding the D2 dopamine receptor (DRD2) is located on human chromosome 11q23 and has been circumstantially associated with a number of human disorders including Parkinson's disease, schizophrenia, and susceptibility to alcoholism. To determine the physical structure of the DRD2 gene, we utilized cosmid cloning, isolation of yeast artificial chromosomes (YACs), and pulsed-field gel electrophoresis to construct a long-range physical map of human chromosome 11q23 linking the genes for the DRD2 and neural cell adhesion molecule (NCAM). The D2 dopamine receptor gene extends over 270 kb and includes an intron of approximately 250 kb separating the putative first exon from the exons encoding the receptor protein. The resulting physical map spans more than 1.5 mb of chromosome band 11q23 and links the DRD2 gene with the gene encoding the NCAM located 150 kb 3' of the DRD2 gene and transcribed from the same DNA strand. We additionally located the sites of at least four hypomethylated HTF islands within the physical map, which potentially indicate the sites of additional genes. High-resolution fluorescent in situ suppression hybridization using cosmid and YAC clones localized this gene cluster between the ApoAI and STMY loci at the interface of bands 11q22.3 and 11q23.1.     

An inactive X specific replication origin associated with a matrix attachment region in the human X linked HPRT gene

Early in female mammalian embryogenesis, one of the two X chromosomes is inactivated to compensate the gene dosage between males and females. One of the features of X chromosome inactivation (XCI) is the late replication of the inactivated X chromosome. This study reports the identification, by competitive PCR of nascent DNA, of a replication origin in intron 2 of the human X-linked HPRT gene, that is functional only on the inactive X. Features frequently associated with replication origins, including a peak of enhanced DNA flexibility, a perfect match to the yeast ACS sequence, a 14/15 match to the Drosophila topoisomerase II consensus, and a 20/21 match to an initiation region consensus sequence, were identified close to the replication origin. The origin is located approximately 2 kb upstream of a matrix attachment region (MAR) and also contains two A:T-rich elements, thought to facilitate DNA unwinding.     

Dispersive initiation of replication in the Chinese hamster rhodopsin locus

Several higher eukaryotic replication origins appear to be composed of broad zones of potential nascent strand start sites, while others are more circumscribed, resembling those of yeast, bacteria, and viruses. The most delocalized origin identified so far is approximately 55 kb in length and lies between the convergently transcribed dihydrofolate reductase (DHFR) and the 2BE2121 genes on chromosome 2 in the Chinese hamster genome. In some of our studies, we have utilized the rhodopsin origin as an early replicating internal standard for assessing the effects of deleting various parts of the DHFR locus on DHFR origin activity. However, it had not been previously established that the rhodopsin locus was located at a site far enough away to be immune to such deletions, nor had the mechanism of initiation at this origin been characterized. In the present study, we have localized the rhodopsin domain to a pair of small metacentric chromosomes and have used neutral/neutral 2-D gel replicon mapping to show that initiation in this origin is also highly delocalized, encompassing a region more than 50 kb in length that includes the nontranscribed rhodopsin gene itself. The initiation zone is flanked at least on one end by an actively transcribed gene that does not support initiation. Thus, the DHFR and rhodopsin origins belong to a class of complex, polydisperse origins that appears to be unique to higher eukaryotic cells.     

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.     

Activity of the c-myc Replicator at an Ectopic Chromosomal Location

DNA replication starts at multiple discrete sites across the human chromosomal c-myc region, including two or more sites within 2.4 kb upstream of the c-myc gene. The corresponding 2.4-kb c-myc origin fragment confers autonomously replicating sequence (ARS) activity on plasmids, which specifically initiate replication in the origin fragment in vitro and in vivo. To test whether the region that displays plasmid replicator activity also acts as a chromosomal replicator, HeLa cell sublines that each contain a single copy of the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanked by selectable markers were constructed. A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfecting a donor plasmid containing the 2.4-kb c-myc origin fragment and FRT, along with a plasmid expressing the yeast FLP recombinase, into cells containing a chromosomal FRT acceptor site. The amount of short nascent DNA strands at the chromosomal acceptor site was quantitated before and after targeted integration of the origin fragment. Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nascent DNA relative to that at the unoccupied acceptor site and to that after the insertion of non-myc DNA. The abundance of nascent strands was greatest close to the c-myc insert of the integrated donor plasmid, and significant increases in nascent strand abundance were observed at sites flanking the insertion. These results provide biochemical and genetic evidence for the existence of chromosomal replicators in metazoan cells and are consistent with the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA.     

A broad replication origin of Drosophila melanogaster, oriD α, consists of AT-rich multiple discrete initiation sites

This work shows that the replication origin of Drosophila melanogaster, oriDalpha, consists of multiple discrete initiation sites. We attempted to map at high resolution the initiation sites in oriDalpha with a quantitative nascent DNA abundance assay using a competitive polymerase chain reaction (PCR) method. Nascent DNA was prepared from either cells blocked in very early S-phase and then labeled with 5-bromo-2'-deoxyuridine (BrdU), or asynchronously growing cells labeled briefly with BrdU. Denatured DNA was size-fractionated in alkaline sucrose gradients. BrdU-labeled nascent DNA was immuno-affinity purified using anti-BrdU antibodies. DNA was quantified with a competitive PCR method before and after immuno-purification. The results indicated that oriDalpha, whose size was presumed to be about 10 kb from two-dimensional gel electrophoretic analysis, contained four major initiation sites in its central 2.8 kb region, and six to approximately eight sites in 8.4 kb. All initiation sites corresponded with AT-rich sequences. Detailed analysis of one major initiation site indicated that its range was restricted to 700 bp.     

Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.     

Multiple sites of replication initiation in the human beta-globin gene locus

The cell cycle-dependent, ordered assembly of protein prereplicative complexes suggests that eukaryotic replication origins determine when genomic replication initiates. By comparison, the factors that determine where replication initiates relative to the sites of prereplicative complex formation are not known. In the human globin gene locus previous work showed that replication initiates at a single site 5′ to the β-globin gene when protein synthesis is inhibited by emetine. The present study has examined the pattern of initiation around the genetically defined β-globin replicator in logarithmically growing HeLa cells, using two PCR-based nascent strand assays. In contrast to the pattern of initiation detected in emetine-treated cells, analysis of the short nascent strands at five positions spanning a 40 kb globin gene region shows that replication initiates at more than one site in non-drug-treated cells. Quantitation of nascent DNA chains confirmed that replication begins at several locations in this domain, including one near the initiation region (IR) identified in emetine-treated cells. However, the abundance of short nascent strands at another initiation site ~20 kb upstream is ~4-fold as great as that at the IR. The latter site abuts an early S phase replicating fragment previously defined at low resolution in logarithmically dividing cells.     

Mechanism of replication of human mitochondrial DNA. Localization of the 5' ends of nascent daughter strands

Human mitochondrial DNA contains two physically separate and distinct origins of DNA replication. The initiation of each strand (heavy and light) occurs at a unique site and elongation proceeds unidirectionally. Animal mitochondrial DNA is novel in that short nascent strands are maintained at one origin (D-loop) in a significant percentage of the molecules. In the case of human mitochondrial DNA, there are three distinct D-loop heavy strands differing in length at the 5' end. We report here the localization of the 5' ends of nascent daughter heavy strands originating from the D-loop region. Analyses of the map positions of 5' ends relative to known restriction endonuclease cleavage sites and 5' end nucleotides indicate that the points of initiation of D-loop synthesis and actual daughter strands are the same. In contrast, the second origin is located two-thirds of the way around the genome where light strand synthesis is presumably initiated on a single-stranded template. Mapping of 5' ends of daughter light strands at this origin relative to known restriction endonuclease cleavage sites reveals two distinct points of initiation separated by 37 nucleotides. This origin is in the same relative genomic position and shows a high degree of DNA sequence homology to that of mouse mitochondrial DNA. In both cases, the DNA region within and immediately flanking the origin of DNA replication contains five tightly clustered tRNA genes. A major portion of the pronounced DNA template secondary structure at this origin includes the known tDNA sequences.     

Functional genomic mapping of an early-activated centromeric mammalian origin of DNA replication.

Ors12, a mammalian autonomously replicating sequence (812 bp), was previously isolated by extrusion of African green monkey (CV-1 cells) nascent DNA from active replication bubbles. It contains a region of alpha-satellite extending 168-bp from the 5'-end, and a nonrepetitive portion extending from nucleotide position 169 to nucleotide 812 that is present in less than nine copies per haploid genome. Ors12 is capable of transient autonomous DNA replication in vivo and in vitro, associates with the nuclear matrix in a cell cycle-dependent manner, and hybridizes at the centromeric region of six CV-1 cell chromosomes as well as a marker chromosome. To demonstrate that DNA replication initiates at ors12 at a native chromosomal locus, a 14.2 kb African green monkey genomic clone was isolated and sequence information was obtained that allowed us to generate eight sets of PCR primers spanning a region of 8 kb containing ors12. One set of primers occurred inside ors12. These primers were used to amplify nascent DNA strands from asynchronously growing CV-1 and African green monkey kidney (AGMK) cells, using noncompetitive and competitive PCR-based mapping methodologies. Both assays showed that DNA replication in vivo initiates preferentially in a 2.3 kb region containing ors12, as well as at a second site located 1.7 kb upstream of ors12. This study provides the first demonstration of genomic function for a centromeric mammalian origin of DNA replication, originally isolated by nascent strand extrusion.     

Initiation of SV40 DNA replication in vivo: location and structure of 5' ends of DNA synthesized in the ori region

Initiation sites for DNA synthesis were located at the resolution of single nucleotides in and about the genetically defined origin of replication (ori) in replicating SV40 DNA purified from virus-infected cells. About 50% of the DNA chains contained an oligoribonucleotide of six to nine residues covalently attached to their 5' ends. Although the RNA-DNA linkage varied, the putative RNA primer began predominantly with rA. The data reveal that initiation of DNA synthesis is promoted at a number of DNA sequences that are asymmetrically arranged with respect to ori: 5' ends of nascent DNA are located at several sites within ori, but only on the strand that also serves as the template for early mRNA, while 5' ends of nascent DNA with the opposite orientation are located only outside ori on its early gene side. This clear transition between discontinuous (initiation sites) and continuous (no initiation sites) DNA synthesis defines the origin of bidirectional replication at nucleotides 5210--5211 and demonstrates that discontinuous synthesis occurs predominantly on the retrograde arms of replication forks. Furthermore, it appears that the first nascent DNA chain is initiated within ori by the same mechanism used to initiate nascent DNA ("Okazaki fragments") throughout the genome.     

Identification of an origin of bidirectional DNA replication in mammalian chromosomes

Mechanistically, an origin of bidirectional DNA replication (OBR) can be defined by the transition from discontinuous to continuous DNA synthesis that must occur on each template strand at the site where replication forks originate. This results from synthesis of Okazaki fragments predominantly on the retrograde arms of forks. We have identified these transitions at a specific site within a 0.45 kb sequence approximately 17 kb downstream from the 3' end of the dihydrofolate reductase gene in Chinese hamster ovary chromosomes. At least 80% of the replication forks in a 27 kb region emanated from this OBR. Thus, initiation of DNA replication in mammalian chromosomes uses the same replication fork mechanism previously described in a variety of prokaryotic and eukaryotic genomes, suggesting that mammalian chromosomes also utilize specific cis-acting sequences as origins of DNA replication.