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PubMed1982

Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication.

We report studies on the mechanism of initiation of DNA replication by p15A, a small plasmid whose origin of replication is known to function much as does that of ColE1. Previous work has shown that an RNA primer for DNA synthesis is generated by the action of RNase H (EC 3.1.26.4) on a precursor transcript. The precursor initiates well upstream of the origin of replication and somehow forms a hybrid with its template during transcription. Here we show that when RNase H cleaves the hybrid at 0 degrees C, an additional cleavage product besides the primer can be identified. Using two-dimensional RNA sequencing techniques, we have established the sequence of this product to within a few nucleotides of each end. The position of the 5' end indicates that the nuclease introduces a nick or very small gap in the precursor at the origin. This suggests that some sequence or structure directs the enzyme to the origin. The position of its 3' end indicates that the precursor terminates at or near a series of six dAs in the template strand about 190 nucleotides from the origin of replication. The data indicate that hybrid formation may be necessary for termination of the precursor at this downstream site.

Base Sequence↗
PubMed1996

An origin of DNA replication from Lactococcus lactis bacteriophage c2.

An origin of DNA relication was identified in the intergenic region between the early and late gene regions of prolate lactococcal phage c2. A DNA fragment containing this origin, designated ori, was shown to direct DNA replication in Lactococcus lactis but not in Escherichia coli. A comparison of ori with the corresponding regions of other prolate phages revealed strict conservation of the nucleotide sequence in one half of this intergenic region. This conserved region alone would not support DNA replication. No open reading frames were identified in the ori fragment, suggesting that host factors alone are sufficient to initiate DNA replication at ori. A novel class of lactococcal vectors and E. coli-L. lactis shuttle vectors based on ori have been constructed.

Base Sequence↗
PubMed1999

Tamplicon-7, a novel T-lymphotropic vector derived from human herpesvirus 7.

We describe the derivation of a novel T-cell-defective virus vector employing the human herpesvirus 7 (HHV-7). The new vector, designated Tamplicon-7, replicates in CD4(+) T cells. The system is composed of a helper virus and defective virus genomes derived by the replication of the input Tamplicon vector. There are two cis-acting functions required for the replication and packaging of the defective virus genomes in the presence of the helper virus: the viral DNA replication origin and the composite cleavage and packaging signal, which directs the cleavage and packaging of defective virus genomes. Viral DNA replication is compatible with the rolling circle mechanism, producing large head-to-tail concatemers of the Tamplicon vector. Thus, in the presence of the helper virus, the replicated vectors are packaged and secreted into the medium. Furthermore, we have shown that the vector can be employed to express a foreign gene, encoding the green fluorescent protein, in the T cells infected with the HHV-7 helper virus. We predict that the Tamplicon-7 vector might be potentially useful for gene therapy of diseases affecting the human CD4(+) T cells, including autoimmune diseases, T-cell lymphomas, and AIDS.

Base Sequence↗
PubMed1996

Identification of a partition and replication region in the Alcaligenes eutrophus megaplasmid pMOL28.

A 4.64 kb region of the 180 kb heavy metal resistance plasmid pMOL28 of Alcaligenes eutrophus CH34, previously shown to be able to replicate autonomously, was sequenced and analyzed. Three genes involved in plasmid maintenance were identified: parA28 and parB28 are involved in plasmid partitioning and stability, while repA28 encodes a protein required for replication. In addition to the par AB28 genes, a third locus, parS28, required in cis active partitioning was identified. The parABS28 locus of pMOL28 shows strong similarity in organization to the sop, par and rep regions, respectively, of the Escherichia coli F-factor, the E.coli P1 and P7 prophages and the Agrobacterium pTiB6S3 and pRiA4b plasmids. The ParAB28 proteins of pMOL28 also show similarity to the proteins encoded by two conserved open reading frames present in the replication regions of the Pseudomonas putida and Bacillus subtilis chromosomes. The functionality of the pMOL28 par region was examined by performing stability and incompatibility tests between pMOL28 and pMOL846 or pMOL850 which contain the 4.64 EcoRI replicon fragment of pMOL28, cloned in opposite orientations into pSUP202, which is itself unable to replicate in A. eutrophus. The RepA2 8 replication protein showed similarity to the RepL protein of P1, which is required for lytic replication of this E. coli phage. The replication origin of pMOL28, oriV28, seems to be located within the repA28 coding region, and pMOL28 replication may depend on transcriptional activation of oriV28.

Alcaligenes↗
PubMed2003

Common domains in the initiators of DNA replication in Bacteria, Archaea and Eukarya: combined structural, functional and phylogenetic perspectives.

Although DNA replication is the universal process for the transmission of genetic information in all living organisms, until very recently evidence was lacking for a related structure and function in the proteins (initiators) that trigger replication in the three 'Life Domains' (Bacteria, Archaea and Eukarya). In this article new data concerning the presence of common features in the initiators of chromosomal replication in bacteria, archaea and eukaryotes are reviewed. Initiators are discussed in the light of: (i) The structure and function of their conserved ATPases Associated with various cellular Activities (AAA+) and winged-helix domains. (ii) The nature of the macromolecular assemblies that they constitute at the replication origins. (iii) Their possible phylogenetic relationship, attempting to sketch the essentials of a hypothetical DNA replication initiator in the micro-organism proposed to be the ancestor of all living cells.

Amino Acid Sequence↗
PubMed2004

Poly(ADP-ribose) polymerase 1 binds to Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat sequence and modulates KSHV replication in latency.

During latency, Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to replicate once and to be partitioned in synchrony with the cell cycle of the host. In this replication cycle, the KSHV terminal repeat (TR) sequence functions as a replication origin, assisted by the latency-associated nuclear antigen (LANA). Thus, TR seems to function as a cis element for the replication and partitioning of the KSHV genome. Viral replication and partitioning are also likely to require cellular factors that interact with TR in either a LANA-dependent or -independent manner. Here, we sought to identify factors that associate with TR by using a TR DNA column and found that poly(ADP-ribose) polymerase 1 (PARP1) and known replication factors, including ORC2, CDC6, and Mcm7, bound to TR. PARP1 bound directly to a specific region within TR independent of LANA, and LANA was poly(ADP-ribosyl)ated by PARP1. Drugs such as hydroxyurea and niacinamide, which raise or lower PARP activity, respectively, affected the virus copy number in infected cells. Thus, the poly(ADP-ribosyl)ation status of LANA appears to affect the replication and/or maintenance of the viral genome. Drugs that specifically up-regulate PARP activity may lead to the disappearance of latent KSHV.

Antigens, Viral↗
PubMed1993

Origin activation and formation of single-strand TG1-3 tails occur sequentially in late S phase on a yeast linear plasmid.

In order to understand the mechanisms leading to the complete duplication of linear eukaryotic chromosomes, the temporal order of the events involved in replication of a 7.5-kb Saccharomyces cerevisiae linear plasmid called YLpFAT10 was determined. Two-dimensional agarose gel electrophoresis was used to map the position of the replication origin and the direction of replication fork movement through the plasmid. Replication began near the center of YLpFAT10 at the site in the 2 microns sequences that corresponds to the 2 microns origin of DNA replication. Replication forks proceeded bidirectionally from the origin to the ends of YLpFAT10. Thus, yeast telomeres do not themselves act as origins of DNA replication. The time of origin utilization on YLpFAT10 and on circular 2 microns DNA in the same cells was determined both by two-dimensional gel electrophoresis and by density transfer experiments. As expected, 2 microns DNA replicated in early S phase. However, replication of YLpFAT10 occurred in late S phase. Thus, the time of activation of the 2 microns origin depended upon its physical context. Density transfer experiments established that the acquisition of telomeric TG1-3 single-strand tails, a predicted intermediate in telomere replication, occurred immediately after the replication forks approached the ends of YLpFAT10. Thus, telomere replication may be the very last step in S phase.

Chromosomes, Fungal↗
PubMedDate not supplied

Single molecule analysis of DNA replication.

We describe here a novel approach for the study of DNA replication. The approach is based on a process called molecular combing and allows for the genome wide analysis of the spatial and temporal organization of replication units and replication origins in a sample of genomic DNA. Molecular combing is a process whereby molecules of DNA are stretched and aligned on a glass surface by the force exerted by a receding air/water interface. Since the stretching occurs in the immediate vicinity of the meniscus, all molecules are identically stretched in a size and sequence independent manner. The application of fluorescence hybridization to combed DNA results in a high resolution (1 to 4 kb) optical mapping that is simple, controlled and reproducible. The ability to comb up to several hundred haploid genomes on a single coverslip allows for a statistically significant number of measurements to be made. Direct labeling of replicating DNA sequences in turn enables origins of DNA replication to be visualized and mapped. These features therefore make molecular combing an attractive tool for genomic studies of DNA replication. In the following, we discuss the application of molecular combing to the study of DNA replication and genome stability.

Animals↗
PubMed1998

Transcription-dependent DNA transactions in the mitochondrial genome of a yeast hypersuppressive petite mutant.

Mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae contains highly conserved sequences, called rep/ori, that are associated with several aspects of its metabolism. These rep/ori sequences confer the transmission advantage exhibited by a class of deletion mutants called hypersuppressive petite mutants. In addition, because they share features with the mitochondrial leading-strand DNA replication origin of mammals, rep/ori sequences have also been proposed to participate in mtDNA replication initiation. Like the mammalian origins, where transcription is used as a priming mechanism for DNA synthesis, yeast rep/ori sequences contain an active promoter. Although transcription is required for maintenance of wild-type mtDNA in yeast, the role of the rep/ori promoter as a cis-acting element involved in the replication of wild-type mtDNA is unclear, since mitochondrial deletion mutants need neither transcription nor a rep/ori sequence to maintain their genome. Similarly, transcription from the rep/ori promoter does not seem to be necessary for biased inheritance of mtDNA. As a step to elucidate the function of the rep/ori promoter, we have attempted to detect transcription-dependent DNA transactions in the mtDNA of a hypersuppressive petite mutant. We have examined the mtDNA of the well-characterized petite mutant a-1/1R/Z1, whose repeat unit shelters the rep/ori sequence ori1, in strains carrying either wild-type or null alleles of the nuclear genes encoding the mitochondrial transcription apparatus. Complex DNA transactions were detected that take place around GC-cluster C, an evolutionarily conserved GC-rich sequence block immediately downstream from the rep/ori promoter. These transactions are strictly dependent upon mitochondrial transcription.

DNA Replication↗
PubMed1999

Difference in strength of autonomously replicating sequences among repeats in the rDNA region of Saccharomyces cerevisiae.

The rDNA region of Saccharomyces cerevisiae contains 100-200 tandemly repeated copies of a 9 kb unit, each with a potential replication origin. In the present studies of cloned fragments from the region involved in the regulation of replication of rDNA, we detected differences in autonomously replicating sequence (ARS) activity for clones from the same yeast strain. One clone, which showed very low ARS activity, carried a point mutation, a C instead of T, in position 9 of the essential 11 bp consensus ARS as compared to clones carrying the normal 10-of-11-bp match to the consensus. The mutation could be traced back to genomic rDNA where it represents about one-third of the rDNA units in that strain. Differences in ARS activity have implications for understanding the regulation of replication of rDNA, and the ratio of active to inactive ARS in the rDNA region may be important for potential generation of extrachromosomal copies.

Base Sequence↗
PubMed2002

14-3-3s are DNA-replication proteins.

14-3-3 proteins are conserved multifunctional molecules, involved in many biological processes. Several 14-3-3 isoforms were recently shown to be cruciform DNA-binding proteins, which is a new activity ascribed to the 14-3-3 family. As cruciform-binding proteins, 14-3-3 proteins are putatively involved in the regulation of DNA replication. Inverted repeat sequences that are able to extrude into cruciform structures are a common feature of replication origins in both prokaryotes and eukaryotes. The involvement of cruciform structures in the initiation of DNA replication has been demonstrated. A leading model of 14-3-3 function proposes that they facilitate critical protein-protein interactions, thus serving as a central component of a wide variety of cellular processes.

14-3-3 Proteins↗
PubMed1998

Initiation and velocity of chromosome replication in Escherichia coli B/r and K-12.

The macromolecular composition and a number of parameters affecting chromosome replication were examined over a range of exponential growth rates in two common Escherichia coli strains, B/r and K-12 AB1157. Based on improved measurements of DNA after treatment of exponential cultures with rifampin, the cell mass per chromosomal replication origin (initiation mass) and the time required to replicate the chromosome from origin to terminus (C period) were determined. For these two strains, the initiation mass approached values of 8 x 10(-10) and 10 x 10(-10) units of optical density (at 460 nm) of culture mass per oriC, respectively, at growth rates above 1 doubling/h (at 37 degrees C). The amount of protein per oriC decreased with increasing growth rate for AB1157 and remained nearly constant for the B/r strain. The C period decreased for both strains in an essentially identical manner from about 70 min at 0.6 doublings/h to about 33 min at 3 doublings/h. From the initiation mass and C period, relative or absolute copy numbers for genes with known map locations can be accurately determined at different growth rates. At growth rates above 2 doublings/h, when chromosomes are highly branched, genes near the origin are about threefold more prevalent than genes near the terminus. At a growth rate of 0.6 doubling/h, this ratio is only about 1.7, which reflects the lower degree of chromosome branching.

Chromosomes, Bacterial↗
PubMed2005

Mechanisms of transcription-replication collisions in bacteria.

While collisions between replication and transcription in bacteria are deemed inevitable, the fine details of the interplay between the two machineries are poorly understood. In this study, we evaluate the effects of transcription on the replication fork progression in vivo, by using electrophoresis analysis of replication intermediates. Studying Escherichia coli plasmids, which carry constitutive or inducible promoters in different orientations relative to the replication origin, we show that the mutual orientation of the two processes determines their mode of interaction. Replication elongation appears not to be affected by transcription proceeding in the codirectional orientation. Head-on transcription, by contrast, leads to severe inhibition of the replication fork progression. Furthermore, we evaluate the mechanism of this inhibition by limiting the area of direct contact between the two machineries. We observe that replication pausing zones coincide exactly with transcribed DNA segments. We conclude, therefore, that the replication fork is most likely attenuated upon direct physical interaction with the head-on transcription machinery.

DNA Replication↗
PubMed2005

Control of ATP-dependent binding of Saccharomyces cerevisiae origin recognition complex to autonomously replicating DNA sequences.

Eukaryotic origin recognition complexes (ORCs) play pivotal roles in the initiation of chromosomal DNA replication. ORC from the yeast, Saccharomyces cerevisiae, recognizes and binds replication origins in the late G1 phase and the binding has profound implications in the progression of the cell cycle to the S-phase. Therefore, we have quantitatively analyzed the mechanism of recognition and interaction of the yeast ORC with various elements of a yeast origin of DNA replication, the autonomously replicating sequence 1 (ARS1). ORC bound all four individual A and B elements of ARS1 with reasonably high affinities. However, the highest affinity binding was observed with a DNA sequence containing both the A and B1 elements. In addition, ATP and ADP significantly modulated the binding of ORC to the combined elements as well as modulating the binding of ORC to the element A alone or in combination with the B1 element. However, binding of ORC to individual B1, B2, and B3 elements was not responsive to nucleotides. Thus, the consensus ARS sequence in element A appeared to play a pivotal role in the ATP-dependent binding of ORC to ARS1 and likely in other ARSs or origins of DNA replication.

Adenosine Diphosphate↗
PubMed1983

Suppressors of a temperature-sensitive copy-number mutation in plasmid NTP1.

A temperature-sensitive high copy-number mutant of plasmid NTP1, first described by Grindley et al. (1978), is lethal to bacterial cells at the non-permissive temperature. This behavior has been used to select mutations in the plasmid replication origin region that suppress the over-replication phenotype. We have identified the site of the original ts lethal mutation and the positions of the reversion mutations. The ts mutation, designated orp, for over-replication, is a single nucleotide change 23 base-pairs upstream from the transcription start site for RNA I, the repressor of plasmid replication. This change simultaneously affects the promoter for RNA I and the precursor of the primer for plasmid replication, RNA II, which is also transcribed from this region. Fusions of the mutant promoter region with the galK gene indicate that transcription is not temperature sensitive. This result suggests that the mutation affects RNA II secondary structure. The reversion mutations are also located within the RNA II coding region more than 200 bp from the site of the original ts mutation. These mutations may also affect RNA II structure.

Escherichia coli↗
PubMed2002

Preformed hexamers of SV40 T antigen are active in RNA and origin-DNA unwinding.

Preformed hexamers of simian virus 40 (SV40) large tumor antigen (T antigen) constitute the bulk of T antigen in infected cells and are stable under physiological conditions. In spite of this they could not be assigned a function in virus replication or transformation. We report that preformed hexamers represent the active T antigen RNA helicase. Monomers and smaller oligomeric forms of T antigen were inactive due to the lack of hexamer formation under RNA unwinding conditions. In contrast to the immunologically related cellular DEAD-box protein p68, the T antigen RNA helicase is found to act in a much more processive way and it does not catalyze rearrangements of structured RNAs. Thereby, it rather seems to resemble other virus-encoded RNA helicases, like vaccinia virus NPH-II. Surprisingly, in our hands preformed hexamers also strikingly bound to and unwound the SV40 replication origin, pointing to a possible role of preformed hexamers in the initiation step of viral DNA replication. Furthermore, we have detected an extra hexamer-specific, high-affinity T antigen ATP binding site with a very slow exchange rate constant, the function of which is discussed.

Adenosine Triphosphate↗
PubMed2002

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein.

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.

Amino Acid Substitution↗
PubMed1989

Analysis of origin of DNA replication of human papovavirus BK.

We have analyzed the cis-acting sequence elements and properties of the origin of DNA replication of human papovavirus BK (BKV). The precise boundaries of the origin varied, depending on the cell type and the viral T antigen used for assay. The BKV minimal origin of replication consisted of an inverted repeat, T-antigen-binding site II, and a 20-base-pair AT block when assayed in monkey kidney CV1 and HeLa cells by using the BKV T antigen. This 76-base-pair minimal origin did not replicate in COS cells in the presence of the simian virus 40 (SV40) T antigen. Unlike that from the SV40 minimal origin, replication from the BKV minimal origin was not enhanced by BKV ori-flanking sequences in CV1 or HeLa cells, using the BKV T antigen. BKV ori-flanking sequences did activate the SV40 minimal origin of replication in COS cells and relieved the orientation-dependent property of this origin. Finally, the BKV T antigen was found to autoregulate activity of the BKV early transcriptional regulatory region. The BKV origin of replication shows similarities to and differences from those of the related viruses SV40 and polyomavirus, suggesting that the proteins involved in the initiation of replication interact with origin sequences differently in these viruses.

Animals↗