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PubMed2006

The functional role of Cdc6 in S-G2/M in mammalian cells.

The Cdc6 protein is required for licensing of replication origins before the onset of DNA replication in eukaryotic cells. Here, we examined whether Cdc6 has other roles in mammalian cell-cycle progression from S to G2/M phase. Using RNA interference, we showed that depletion of Cdc6 in synchronous G1 cells blocks G1 to S transition, confirming the essential role of Cdc6 in the initiation of DNA replication. In contrast, depletion of Cdc6 in synchronous S-phase cells slowed DNA replication and led to mitotic lethality. The Cdc6-depleted S-phase cells showed fewer newly fired origins; however, established replication forks remained active, even during chromatin condensation. Despite such DNA replication abnormalities, loss of Cdc6 failed to activate Chk1 kinase. These results show that Cdc6 is not only required for G1 origin licensing, but is also crucial for proper S-phase DNA replication that is essential for DNA segregation during mitosis.

Cell Cycle Proteins↗
PubMed1982

Replication origins are associated with transcription initiation sequences in the mitochondrial genome of yeast.

Mitochondrial transcripts have been investigated in a series of spontaneous petite mutants of Saccharomyces cerevisiae endowed with mitochondrial genomes formed by short repeat units containing no genes, but either: (a) one of the seven ori sequences, the canonical origins of DNA replication (ori+ mutants); or (b) partially deleted ori sequences, lacking GC-rich clusters A or C (ori- mutants); or (c) no canonical ori sequence, but only oris sequences, the surrogate origins of replication ( orio mutants). The results indicate that some ori sequences play a role in transcription initiation, and that the presence of cluster C and, more specifically, of an AT-rich sequence next to it, are essential for transcription to take place. Hybridization experiments with separated DNA strands have identified the template strand used in transcription as the strand containing the oligopyrimidine stretch of cluster C. S1 degradation of RNA-DNA hybrids indicated that transcription initiates at a TATTACTTATATATTT sequence next to the oligopyrimidine stretch of cluster C and proceeds in the cluster C----cluster A direction. The relevance of these results for the transcription of the wild-type mitochondrial genome is discussed.

Base Sequence↗
PubMed2002

Sequence-independent DNA binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.

The DnaA protein specifically binds to the origin of chromosomal DNA replication and initiates DNA synthesis. In addition to this sequence-specific DNA binding, DnaA protein binds to DNA in a sequence-independent manner. We here compared the two DNA binding activities. Binding of ATP and ADP to DnaA inhibited the sequence-independent DNA binding, but not sequence-specific binding. Sequence-independent DNA binding, but not sequence-specific binding, required incubation at high temperatures. Mutations in the C-terminal domain affected the sequence-independent DNA binding activity less drastically than they did the sequence-specific binding. On the other hand, the mutant DnaA433, which has mutations in a membrane-binding domain (K327 to I344) was inert for sequence-independent binding, but could bind specifically to DNA. These results suggest that the two DNA binding activities involve different domains and perform different functions from each other in Escherichia coli cells.

Adenine Nucleotides↗
PubMed2001

Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing.

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

Alkaloids↗
PubMed1991

Altered DNA binding and replication activities of JC virus T-antigen mutants.

Ten mutations were introduced into the JC virus (JCV) T antigen within a region corresponding to the SV40 T-antigen DNA binding domain (SV40 amino acids 131 to 220); nine of these increased homology between the two proteins in sequences critical for SV40 T antigen DNA binding. All mutant JCV T antigens bound to JCV and SV40 origins of DNA replication. Binding efficiency relative to the of wild-type JCV T antigen ranged from 83 to 301% for the JCV binding sites and from 44 to 240% for the SV40 binding sites. Nine mutant proteins promoted viral DNA replication in primary human fetal glial (PHFG) and CV-1 cells. In PHFG cells, promotion of DNA replication ranged from 26 to 220% relative to that of wild-type T antigen; in CV-1 cells it ranged from 14 to 522%. Coding sequences for five mutant proteins were transferred into the hybrid virus M1 (SV40) [M1(SV40) contains coding sequences from JCV and regulatory sequences from SV40]. Wild-type T antigen promoted replication weakly from the SV40 origin in these hybrid viruses in CV-1 cells (2% that from the JCV origin); replication driven by the mutant proteins ranged from 110 to 412% of that induced by the wild-type protein. Efficient specific DNA binding by a mutant T antigen was not a reliable indicator of that mutant protein's ability to promote DNA replication.

Amino Acid Sequence↗
PubMed2004

Simian virus 40-based replication of catalytically inactive human immunodeficiency virus type 1 integrase mutants in nonpermissive T cells and monocyte-derived macrophages.

Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the nef position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.

Antigens, Polyomavirus Transforming↗
PubMed1992

Structure of the ColE1 DNA molecule before segregation to daughter molecules.

The segregation of daughter DNA molecules at the end stage of replication of plasmid ColE1 was examined. When circular ColE1 DNA replicates in a cell extract at a high KCl concentration (140 mM), a unique class of molecules accumulates. When the molecule is cleaved by a restriction enzyme that cuts the ColE1 DNA at a single site, an X-shaped molecule in which two linear components are held together around the origin of DNA replication is made. For a large fraction of these molecules, the 5' end of the leading strand remains at the origin and the 3' end of the strand is about 30 nucleotides upstream of the origin. The 3' end of the lagging strand is located at the terH site (17 nucleotides upstream of the origin) and the 5' end of the strand is a few hundred nucleotides upstream of the terH site. Thus the parental strands of the molecule intertwine with each other only once. When the KCl concentration is lowered to 70 mM, practically all of these molecules are converted to daughter circular monomers or to catenanes consisting of two singly interlocked circular units.

Bacterial Proteins↗
PubMed1991

A. E. Braunstein Plenary Lecture. Nuclear skeleton, DNA domains and control of replication and transcription.

Chromosomal DNA is organized in loops or domains of about 100 kb. Their ends seem to be attached to special protein skeletal structures. The DNA-attachment sites can be subdivided into permanent and transient types. The permanent or constitutive attachment sites, which are retained in all types of cells (including those inactive in replication and transcription), either coincide with or are located close to replication origins. This observation provides a simple way for isolation of DNA fragments containing replication origins. Such fragments from the chicken alpha-globin gene domain and other regions of the chicken genome contain DNA sequences which interact with nuclear proteins present in dividing cells, but absent from non-dividing cells. Several new consensus sequences interacting with nuclear proteins were detected. The 5' end region of the alpha-globin gene domain containing a replication origin was found to possess enhancer activity lacking tissue specificity. Hence, the domain organization of DNA is related to the organization of replication process. Other sets of data indicate that the integrity of DNA domains is important for maintaining transcription within the domain. According to these data, even a single nick at an distance of about 100 kbp seems to be sufficient for blocking transcription within the whole domain at the stage of RNA elongation. Thus, topological integrity of DNA may be an important factor involved in formation of active chromatin.

Animals↗
PubMed1998

The DNA replication fork in eukaryotic cells.

Replication of the two template strands at eukaryotic cell DNA replication forks is a highly coordinated process that ensures accurate and efficient genome duplication. Biochemical studies, principally of plasmid DNAs containing the Simian Virus 40 origin of DNA replication, and yeast genetic studies have uncovered the fundamental mechanisms of replication fork progression. At least two different DNA polymerases, a single-stranded DNA-binding protein, a clamp-loading complex, and a polymerase clamp combine to replicate DNA. Okazaki fragment synthesis involves a DNA polymerase-switching mechanism, and maturation occurs by the recruitment of specific nucleases, a helicase, and a ligase. The process of DNA replication is also coupled to cell-cycle progression and to DNA repair to maintain genome integrity.

Cell Cycle↗
PubMed2006

Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli.

A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct, a unit-length chimeric PCV1(Rep)/PCV2(Cap) genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1(Cap)/PCV2(Rep) genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE(1) Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep' protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep' protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.

Animals↗
PubMed1992

ORS12, a mammalian autonomously replicating DNA sequence, is present at the centromere of CV-1 cell chromosomes.

ors12, an 812-bp-long sequence, previously isolated by extrusion of nascent DNA from replication bubbles active at the onset of S phase (G. Kaufmann, M. Zannis-Hadzopoulus, and R. G. Martin Mol. Cell. Biol. 5, 721-727, 1985), has been shown to function as an origin of DNA replication in autonomously replicating plasmids (L. Frappier and M. Zannis-Hadjopoulos Proc. Natl. Acad. Sci. USA 84, 6668-6672, 1987) and in a cell-free system (C. E. Pearson, L. Frappier, and M. Zannis-Hadzopoulos Biochim. Biophys. Acta 1090, 156-166, 1991). A portion of ors12 (nucleotides 1-168) consists of the highly reiterated alpha-satellite sequence (B. S. Rao et al. Gene 87, 233-242, 1990). We have estimated the copy number of the non-alpha-satellite portion of ors12 in CV-1 cells to be < 9 copies per haploid genome and have used it as a probe to generate a genomic map of ors12 on CV-1 DNA. In situ hybridization of CV-1 metaphase chromosomes, using a biotinylated probe of the entire ors12 sequence, positively identified the centromeres of all chromosomes. However, when the non-alpha-satellite portion of ors12 was used as a probe, it positively identified the centromeric region of only six chromosomes, namely, B4, C11, D14, D24, E25, and E27, as well as that of a marker chromosome. The results suggest that ors12 represents a centromeric putative replication origin that is present on a subset of CV-1 chromosomes and is activated at the onset of S phase.

Animals↗
PubMed2006

The origin of viruses and their possible roles in major evolutionary transitions.

Viruses infecting cells from the three domains of life, Archaea, Bacteria and Eukarya, share homologous features, suggesting that viruses originated very early in the evolution of life. The three current hypotheses for virus origin, e.g. the virus first, the escape and the reduction hypotheses are revisited in this new framework. Theoretical considerations suggest that RNA viruses may have originated in the nucleoprotein world by escape or reduction from RNA-cells, whereas DNA viruses (at least some of them) might have evolved directly from RNA viruses. The antiquity of viruses can explain why most viral proteins have no cellular homologues or only distantly related ones. Viral proteins have replaced the ancestral bacterial RNA/DNA polymerases and primase during mitochondrial evolution. It has been suggested that replacement of cellular proteins by viral ones also occurred in early evolution of the DNA replication apparatus and/or that some DNA replication proteins originated directly in the virosphere and were later on transferred to cellular organisms. According to these new hypotheses, viruses played a critical role in major evolutionary transitions, such as the invention of DNA and DNA replication mechanisms, the formation of the three domains of life, or else, the origin of the eukaryotic nucleus.

Amino Acid Sequence↗
PubMed1990

Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding.

By mutational analysis, we have identified a motif critical to the proper recognition and binding of simian virus 40 large tumor antigen (T antigen) to virus DNA sequences at the origin of DNA replication. This motif is tripartite and consists of two elements (termed A1 and B2) that are necessary for sequence-specific binding of the origin and a central element (B1) which is required for nonspecific DNA-binding activity. Certain amino acids in elements A1 (residues 152 to 155) and B2 (203 to 207) may make direct contact with the GAGGC pentanucleotide sequences in binding sites I and II on the DNA. Alternatively, these two elements could determine the proper structure of the DNA-binding domain, although for a number of reasons we favor the first possibility. In contrast, element B1 (183 to 187) is most likely important for recognizing a general structural feature of DNA. Elements A1 and B2 are nearly identical in all known papovavirus T antigens, whereas B1 is identical only in the closely related papovaviruses simian virus 40, BK virus, and JC virus. In addition to these three elements, a fourth (B3; residues 215 to 219) is necessary for the binding of T antigen to site II but not to site I. We propose that additional contact sites on T antigen are involved in the interaction with site II to initiate the replication of the viral DNA.

Adenoviruses, Human↗
PubMed1997

Absence of an unusual "densely methylated island" at the hamster dhfr ori-beta.

An unusual "densely methylated island" (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has been reported at mammalian origins of DNA replication. This report had far-reaching implications in understanding of DNA methylation and DNA replication. For example, since this DMI appeared in about 90% of proliferating cells, but not in stationary cells, it may regulate origin activation. In an effort to confirm and extend these observations, the DMI at the well characterized ori-beta locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was checked for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cells and in cell populations enriched in the G1, S, or G2 + M phases of their cell division cycle. DNA was subjected to 1) cleavage by methylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate modification of 5-methylcytosines (mC) followed by piperidine cleavage. The permanganate reaction is a novel method for direct detection of mC residues that complements the more commonly used hydrazine method. These methods were capable of detecting mC in 2% of the cells. At the region of the proposed DMI, only one mC at a CpG site was detected. However, the ori-beta DMI was not detected in any of these cell populations using any of these methods.

Animals↗
PubMed1998

CDC16 controls initiation at chromosome replication origins.

The Cdc28p cyclin-dependent kinase is thought to both catalyze the onset of DNA replication and prevent rereplication by blocking the reassembly of initiation complexes at replication origins. Budding yeast with mutations in the CDC16 gene represent an exception to this model, because they rereplicate DNA despite being in a G2-like arrest with continually elevated Cdc28p kinase activity. We show, in contradiction to Pichler et al. (1997), that the extra DNA that accumulates in cdc16 mutants is largely chromosomal, as we originally reported. Two-dimensional DNA electrophoresis shows that cdc16 mutants reinitiate DNA synthesis from normal chromosome replication origins, and density transfer experiments show that multiple chromosomal locations are affected. Rereplication from origins requires both Cdc6p and Cdc46/Mcm5p, initiation proteins that had been thought to be inactivated by the Cdc28p kinase. These results establish that CDC16 is required to prevent inappropriate firing of replication origins.

Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome↗
PubMed1984

Gene A protein interacting with recombinant plasmid DNAs containing 25-30 b.p. of the phi X174 replication origin.

Synthetic oligodeoxyribonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the 30 b.p. origin region of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the kanamycin resistance gene of pACYC177 (AmpR, KmR). Transformants were picked up by antibiotic selection and filter-hybridization using one of the oligodeoxyribonucleotides as a probe. Approximate lengths of the inserts were determined by restriction enzyme analysis. Exact length and orientation of each insert was determined by DNA sequencing. Plasmid DNA with an insert homologous to the first 25 b.p. of the phi X174 origin is not nicked by the gene A protein. However, plasmid DNA containing the 27 b.p. fragment in either orientation is nicked by the gene A protein, as well as plasmid DNAs containing the first 28 b.p. or the complete 30 b.p. conserved origin region of the isometric phages.

Bacteriophage phi X 174↗
PubMed1988

Minimal region necessary for autonomous replication of pTAR.

The native 44-kilobase-pair plasmid pTAR, discovered in a grapevine strain of Agrobacterium tumefaciens, contains a single origin of DNA replication confined to a 1.0-kilobase-pair region of the macromolecule. This region (ori) confers functions sufficient for replication in Agrobacterium and Rhizobium species but not in Pseudomonas solanacearum, Pseudomonas glumae, Pseudomonas syringae pv. savastanoi, Xanthomonas campestris pv. campestris, and Escherichia coli. ori contains a repA gene that encodes a 28,000-dalton protein required for replication. Nucleotide sequencing of repA and its promoter region revealed four 8-base-pair palindromic repeats upstream of the repA coding region. Deletion of these repeats alters repA expression and plasmid copy number. Downstream of repA are three additional repeats in a region essential for replication. A locus responsible for plasmid partitioning (parA) and a putative second locus regulating plasmid copy number are part of the origin region and are required for stable plasmid maintenance.

Amino Acid Sequence↗