Cofractionation of hela cell replication proteins with Ors-binding activity

Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases α and δ, topoisomerase II, and replication protein A, (RP-A).     

The nucleotide sequence of a DNA fragment from the replication origin of the antibiotic resistance factor R1drd19

Summary The recombinant plasmid pRK101 contains a DNA fragment which carries the complete replication origin of the antibiotic resistance factor R1drd-19 inserted into the vector plasmid pBR322. In a spontaneously arising mutant of this plasmid (pRK 103) a deletion of about 215 base pairs (bp) has been detected by heteroduplex analysis and mapping with restriction endonucleases. Essential parts of the replication origin must be located in the deleted sequence. The deletion mutant pRK103, in contrast to its parent plasmid pRK101 is not replicated under the control of the R1 replicon, even when the R1 factor or copy mutants of it are present within the same cell. These latter plasmids can complement a plasmid-specific protein not coded by pRK101 but essential for R1-directed replication. The nucleotide sequence of a 252 bp HpaII fragment covering about 170–200 bp of the deletion was determined. This piece of DNA is rich in G and C and contains a series of small palindromes, symmetrically arranged repeated sequences and short selfcomplementary structures which may be of significance for the initiation of the DNA replication. The possibility that the sequenced DNA fragment comprises a major part of the replication origin of R1drd-19 is discussed.     

OBA/Ku86: DNA binding specificity and involvement in mammalian DNA replication

Ors-binding activity (OBA) was previously semipurified from HeLa cells through its ability to interact specifically with the 186-basepair (bp) minimal replication origin of ors8 and support ors8 replication in vitro. Here, through competition band-shift analyses, using as competitors various subfragments of the 186-bp minimal ori, we identified an internal region of 59 bp that competed for OBA binding as efficiently as the full 186-bp fragment. The 59-bp fragment has homology to a 36-bp sequence (A3/4) generated by comparing various mammalian replication origins, including the ors. A3/4 is, by itself, capable of competing most efficiently for OBA binding to the 186-bp fragment. Band-shift elution of the A3/4-OBA complex, followed by Southwestern analysis using the A3/4 sequence as probe, revealed a major band of approximately 92 kDa involved in the DNA binding activity of OBA. Microsequencing analysis revealed that the 92-kDa polypeptide is identical to the 86-kDa subunit of human Ku antigen. The affinity-purified OBA fraction obtained using an A3/4 affinity column also contained the 70-kDa subunit of Ku and the DNA-dependent protein kinase catalytic subunit. In vitro DNA replication experiments in the presence of A3/4 oligonucleotide or anti-Ku70 and anti-Ku86 antibodies implicate Ku in mammalian DNA replication.     

Classes of autonomously replicating sequences are found among early-replicating monkey DNA

Thirteen new independent clones of origin-enriched sequences (ors) that are capable of autonomous replication have been identified from a library of 100 ors i clones that had been previously isolated from early replicating monkey (CV-1) DNA. Autonomous replication was assayed by transient episomal replication in transfected HeLa cells; ors-plasmid DNA was isolated at various times after transfection and screened by the DpnI resistance assay and the bromodeoxyuridine (BrdUrd) substitution assay to differentiate between input and newly replicated DNA. Four of the autonomously replicating clones were identified by screening the ors-library with probes of ors 3, 8, 9 and 12, previously shown to be capable of autonomous replication (Frappier and Zannis-Hadjopoulos, Proc. Natl. Acad. Sci. USA (1987) 84, 6668-6672). The other nine functional ors clones were identified among 18 randomly chosen ones, which were similarly screened for autonomous replication. Nucleotide sequence analyses of 11 of the newly identified functional ors plasmids revealed, in most of them, features similar to those present in other viral or eukaryotic replication origins, notably the presence of AT-rich regions and inverted repeats. Pairwise comparisons between the newly identified ors showed no extensive sequence homologies, other than the presence of the alpha-satellite repetitive sequence family in three ors and of the repetitive Alu sequence family in one ors. The results suggest that there exist different classes of mammalian replication origin, highly or moderately repetitive and unique, and that their activation is most probably dependent on the presence of structural determinants rather than on a particular sequence.     

SV40 deletion mutants lacking the 21-bp repeated sequences are viable, but have noncomplementable deficiencies.

We have constructed deletion mutants of simian virus 40 (SV40) lacking the two tandemly repeated copies or all three copies of the 21-bp repeated sequence located in the origin region. The mutants were viable, but had lower infectivities compared to the wild type. The mutant lacking two copies of the 21-bp repeat grew fairly well indicating that the one copy of the 21-bp repeat it contains is adequate. The other mutant lacking all the three copies of the 21-bp repeat was also viable but grew poorly. The viability of this mutant suggests that the upstream 72-bp repeated sequence compensates, though only partially, for the absence of the 21-bp repeat. The growth deficiencies of the deletion mutants could not be overcome by complementation with temperature-sensitive helper mutants providing either the early or the late functions of the virus, suggesting that the deficiencies lie in both early and late gene expression and/or in replication.     

Identification and functional analysis of a human homologue of the monkey replication origin ors8

We previously isolated from African green monkey (CV-1) cells a replication origin, ors8, that is active at the onset of S-phase. Here, its homologous sequence (hors8, accession number: DQ230978) was amplified from human cells, using the monkey-ors8-specific primers. Sequence alignment between the monkey and the human fragment revealed a 92% identity. Nascent DNA abundance analysis, involving quantification by real-time PCR, indicated that hors8 is an active replication origin, as the abundance of nascent DNA from a genomic region containing it was 97-fold higher relative to a non-origin region in the same locus. Furthermore, the data showed that the hors8 fragment is capable of supporting the episomal replication of its plasmid, when cloned into pBlueScript (pBS), as assayed by the DpnI resistance assay after transfection of HeLa cells. A quantitative chromatin immunoprecipitation (ChIP) assay, using antibodies against Ku, Orc2, and Cdc6, showed that these DNA replication initiator proteins were associated in vivo with the human ors8 (hors8). Finally, nascent DNA abundance experiments from human cells synchronized at different phases of the cell cycle revealed that hors8 is a late-firing origin of DNA replication, having the highest activity 8 h after release from late G(1).     

Genomic organization of two families of highly repeated nuclear DNA sequences of maize selected for autonomous replicating activity in yeast

Maize nuclear DNA sequences capable of promoting the autonomous replication of plasmids in yeast were isolated by ligating Eco RI-digested fragments into yeast vectors unable to replicate autonomously. Three such autonomously replicating sequences (ARS), representing two families of highly repeated sequences within the maize genome, were isolated and characterized. Each repetitive family shows hybridization patterns on a Southern blot characteristic of a dispersed sequence. Unlike most repetitive sequences in maize, both ARS families have a constant copy number and characteristic genomic hybridization pattern in the inbred lines examined. Larger genome clones with sequence homology to the ARS-containing elements were selected from a lambda library of maize genomic DNA. There was typically only one copy of an ARS-homologous sequence on each 12–15 kb genomic fragment.     

Replication Protein RepN Encoded by the RC Plasmid of Thermophilic Bacterium Thermoanaerobacterium saccharolyticum: Mutation Analysis and Deletion Mapping of Domains Responsible for Its Lethal Effect

Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110. Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein. Its lethal effect on bacterial cells was unusually high for a protein of the kind. Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity. On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein. The RepN fragments essential for the lethal effect were found to share a motif without appreciable homology to known conserved motifs. The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for stable maintenance of the AT-rich plasmid in thermophilic bacterial cells.     

Site-specific deletion at the replication origin of the antibiotic resistance factor R1

Summary The recombinant plasmid pRK101 carrying the complete replication origin of the antibiotic resistance factor R1 suffers frequently a deletion of 218 base pairs, removing parts or all of the origin sequence. This deletion seems to occur always when the Pst-E fragment carrying the replication origin is inserted into the cloning vector pBR322 in an orientation where the direction of R1 replication is the same as that of the vector plasmid and frequently when it is inserted in the opposite direction. DNA sequence analysis around the junction site generated by the deletion in three independently isolated deletion mutants reveals that the deletion occurs at a specific site, namely the end of a 22 bp sequence which is repeated almost identically at the other end of a segment of 197 bp. During the deletion one repeat unit is removed whereas the other is retained. The DNA sequence included by the two repeats contains high symmetric structures, i.e. inverted repeats, direct repeats and palindromes which may represent regulatory sites of the origin.     

Identification and sequence analysis of the replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503

Abstract The replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503 was localised to within a 10-kb Hin dIII restriction fragment. A 6.3-kb Bgl II- Hin dIII subclone of this fragment, cloned into a replication probe vector, allowed replication in Lactococcus but not in Bacillus or Lactobacillus . Sequence analysis revealed an ORF of 1152 bp preceded by a putative ori region containing a 22-bp sequence tandemly repeated three and three-quarter times, a second smaller direct repeat and two inverted repeats. Extensive homology was observed with the well characterised replication region of the small cryptic plasmid pCI305 (Hayes, F., Vos, P., Fitzgerald, G.F., de Vos, W. and Daly, C. Plasmid 25, 16–26).     

G4 motifs affect origin positioning and efficiency in two vertebrate replicators

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome‐wide studies in vertebrates have recently identified a consensus G‐rich motif potentially able to form G‐quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200‐bp cis‐regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.     

Cloning and Identification of anAgrobacterium radiobacter5D-1 Chromosome Fragment Involved in Controlling Nitrogen Metabolism, Biosynthesis of Indolylacetic Acid, and Replication of ColE1 Plasmids

Pleiotropic chromosomal mutations were earlier identified in saprophytic associative bacterium Agrobacterium radiobacter5D-1. The mutations changed nitrogen metabolism, disturbed synthesis of indolylacetic acid (IAA), and conferred the ability to sustain replication of ColE1 plasmid derivatives, which are not normally maintained in bacteria other than Escherichia.The mutations were designated Nr(Nitrogen metabolism) and assigned to a single cluster on an A. radiobactergenetic map. A 420-bp fragment AGH23.1.1 was cloned from an agrobacterial genomic library. Introduced in the Nr mutants as a part of a pUC18-based recombinant plasmid, the AGH23.1.1 fragment complemented the Nrmutations with respect to nitrogen metabolism and IAA biosynthesis, but transformants still sustained replication of ColE1 plasmids. Transformation with the linear AGH23.1.1 fragment was due to substitution of a mutant allele of the nrgene with its wild-type counterpart as a result of recombination and completely restored the wild phenotype in the Nr mutants, including the inability to maintain ColE1 plasmids. The AGH23.1.1 fragment and its flanking regions were sequenced. The established sequence was shown to contain two open reading frames (ORFs) coding for proteins with unknown functions. Thus, the cloned fragment contained a gene(s) that controls nitrogen metabolism and IAA synthesis and replication of ColE1 plasmids inA. radiobactercells. Possible variants of the genetic control of these processes are considered.     

Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.     

Despite its high representation in extrachromosomal circular DNAs from Drosophila embryos,the dodecasatellite does not allow autonomous replication in cultured cells

Summary— The dodecasatellite is a 11/12 bp tandemly repeated sequence which is overrepresented, with regard to its genomic representation, in extrachromosomal circular DNAs from D melanogaster embryos. Here we show that a bacterial plasmid carrying a cluster of dodecasatellite is not able to replicate efficiently in cultured cells. This observation does not support the hypothesis that the over-representati results from an autonomous replication of dodecasatellite circular DNA molecules.     

Effect of the deletion of a fragment dispensable for the autonomous maintenance of plasmid pT181 on the competition between incompatible plasmids

The deletion of the 560-bp HindIII C fragment from pT181 derivatives does not change the stability or copy number of the plasmid but affects its ability to compete with undeleted, incompatible plasmids for maintenance in the host cell. The disadvantage of the deleted plasmids seems to be manifested at the level of replication. It results that for plasmid pT181 a sequence dispensable for autonomous maintenance and replication control could affect the outcome of the competition between autonomous, incompatible plasmids.     

pHP489, a Helicobacter pylori small cryptic plasmid, harbors a novel gene coding for a replication initiation protein

We have analyzed a Helicobacter pylori plasmid, pHP489. The 1222-bp nucleotide sequence had one open reading frame, a DnaA-binding site, one direct repeat, and three inverted repeats. The (G+C) content of pHP489 was 33.3%. Although the nucleic acid sequence and deduced amino acid sequence were homologous to those of other bacterial plasmid Rep proteins, the degree of similarity was very low. A deletion analysis showed that the Rep protein was not required for the replication of pHP489 in its H. pylori host, but the host replication machinery was needed.     

Cloning and characterization of G+C-rich, highly repeated DNA sequences from Porphyra yezoensis (laver), Rhodophyta

G+C-rich sequences in the genomic DNA of Porphyrayezoensis (laver) were cloned and characterized. Sequence analyses of the genomic DNA inserted in fiveclones showed that the DNA contained long G+C-richstretches of more than 200 bp. These stretchesconsisted of more than 80% G+C residues. TheG+C-rich sequences were highly repeated andinterspersed throughout the genome of P.yezoensis and constituted about 6.0–6.6% of thegenome. Parts of these sequences were tandem repeatedin arrays. Hybridization experiments showed thatthese highly repeated, interspersed G+C-rich sequenceswere present in other species of Porphyra, butnot in species of the genera Grateloupia and Gelidium, suggesting that these sequences mightevolve rapidly among genomes, species and genera.