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PubMed1991

The repression of the reverse-oriented transcription from the adenovirus terminus by NFI in competition with TFIID.

Nuclear factor I (NF) represses the transcription which is promoted by the cloned adenovirus (Ad) type 5 DNA replication origin and is reverse-oriented with respect to the direction of the replication. The mechanism of this repression by NFI was investigated. In the cell-free transcription system, the repression was observed only when NFI was present during the formation of the transcription initiation complex. From the results of DNase I protection experiments, it was indicated that NFI bound to its binding site in the Ad replication origin prevents TFIID from proper binding to the adjacent AT-rich region and consequently represses the transcription.

Adenoviridae↗

PubMed1986

Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA.

Newly replicated herpes simplex virus (HSV) DNA consists of head-to-tail concatemers which are cleaved to generate unit-length genomes bounded by the terminally reiterated a sequence. Constructed defective HSV vectors (amplicons) containing a viral DNA replication origin and the a sequence are similarly replicated into large concatemers which are cleaved at a sequences punctuating the junctions between adjacent repeat units, concurrent with the packaging of viral DNA into nucleocapsids. In the present study we tested the ability of seed amplicons containing specific deletions in the a sequence to become cleaved and packaged and hence be propagated in virus stocks. These studies revealed that two separate signals, located within the Ub and Uc elements of the a sequence, were essential for amplicon propagation. No derivative defective genomes were recovered from seed constructs which lacked the Uc signal. In contrast, propagation of seed constructs lacking the Ub signal resulted in the selection of defective genomes with novel junctions, containing specific insertions of a sequences derived from the helper virus DNA. Comparison of published sequences of concatemeric junctions of several herpesviruses supported a uniform mechanism for the cleavage-packaging process, involving the measurement from two highly conserved blocks of sequences (pac-1 and pac-2) which were homologous to the required Uc and Ub sequences. These results form the basis for general models for the mechanism of cleavage-packaging of herpesvirus DNA.

Animals↗

PubMed1989

Host range bias of the JC virus mutant enhancer with DNA rearrangement.

JC-HEK, a JC virus (JCV) host range mutant adapted to growth in human embryonic kidney cells (HEK), has DNA rearrangement in the control region for early transcription. The 822-bp rearranged segment of JC-HEK (containing one authentic promoter-enhancer unit of 98 bp and truncated coding region of T-antigen and VP-1 genes) was inserted into pSV0cat vector and examined for expression of chloramphenicol acetyltransferase (CAT). The CAT activity induced by the mutant was comparable to that by the SV40 promoter-enhancer (pSV2cat) and four times as high as that by the prototype JCV (Mad-1) in HEK cells, whereas the two JCV promoter-enhancers were equally efficient in a permissive neuroblastoma cell line. On the other hand, both JC-HEK DNA with three DNA replication origins and Mad-1 DNAs replicated after transfection into HEK cells at a similar efficiency in the presence of JCV T-antigen. The elevated promotor-enhancer activity caused by the DNA rearrangement seems to play a major role in adaptation of JCV to growth in HEK cells.

Antigens, Polyomavirus Transforming↗

PubMed1980

Physical mapping of primary and secondary origins of bacteriophage T7 DNA replication.

Deletion mutants of bacteriophage T7 have been used to identify and to map, by electron microscopy, the origins of T7 DNA replication. The primary origin of phage T7 DNA replication lies within a 100-base-pair region located 14.75-15.0% of the distance from the genetic left end of the DNA molecule. T7 phage whose DNA contains a deletion of this region initiate replication at secondary origins, the predominant one of which is located at a distance approximately 4% from the left end of the molecule.

Chromosome Deletion↗

PubMed1994

Activation of a yeast replication origin near a double-stranded DNA break.

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.

DNA Damage↗

PubMed1995

RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA.

RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion.

Animals↗

PubMed2004

Changes in the structure of DNA molecules and the amount of DNA per plastid during chloroplast development in maize.

We examined the DNA from chloroplasts obtained from different tissues of juvenile maize seedlings (from eight to 16 days old) and adult plants (50-58 days old). During plastid development, we found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules. The decrease in molecular size and complexity of the DNA paralleled a progressive decrease in DNA content per plastid. Most surprising, we were unable to detect DNA of any size in most chloroplasts from mature leaves, long before the onset of leaf senescence. Thus, the DNA content per plastid is not constant but varies during development from hundreds of genome copies in the proplastid to undetectable levels in the mature chloroplast. This loss of DNA from isolated, mature chloroplasts was monitored by three independent methods: staining intact chloroplasts with 4',6-diamidino-2-phenylindole (DAPI); staining at the single-molecule level with ethidium bromide after exhaustive deproteinization of lysed chloroplasts; and blot-hybridization after standard DNA isolation procedures. We propose a mechanism for the production of multigenomic chloroplast chromosomes that begins at paired DNA replication origins on linear molecules to generate a head-to-tail linear concatemer, followed by recombination-dependent replication.

Bacteriophage T4↗

PubMed1987

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.

Alleles↗

PubMed1981

Simian virus 40 tandem repeated sequences as an element of the early promoter.

On the late side of the simian virus 40 (SV40) DNA replication origin are several sets of tandem repeated sequences, the largest of which is 72 base pairs long. The role of these sequences was examined through construction of deletion mutants of SV40. A mutant from which one of the 72-base-pair repeated units was removed is viable upon transfection of monkey kidney cells with viral DNA. Extension of this deletion into the second repeated unit, however, leads to nonviability, as recognized by the absence of early transcription and of tumor antigen production. These observations indicate that the 72-base-pair repeated sequences form an essential element in the early viral transcriptional promoter and explain the inability of such a deleted genome to complement an early temperature-sensitive mutant of SV40, tsA, as well as the failure to replicate its DNA. In a parallel experiment it was found that the extended deletion mutant was also unable to complement a late temperature-sensitive mutant of SV40, tsB. This suggests that the extended mutant is also defective in DNA replication or late transcription (or both).

Base Sequence↗

PubMed1985

Adenovirus sequences required for replication in vivo.

We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occupies the first 18 to 21 bp and includes sequences conserved between all adenovirus serotypes. The adjacent auxillary region extends past nucleotide 36 but not past nucleotide 67 and contains the binding site for nuclear factor I.

Adenoviridae↗

PubMed1985

Differential activation of hybrid genes containing herpes simplex virus immediate-early or delayed-early promoters after superinfection of stable DNA-transfected cell lines.

We compared the levels of gene expression obtained after herpes simplex virus (HSV) superinfection of cell lines containing integrated human beta-interferon (IFN) or chloramphenicol acetyltransferase (CAT) genes under the control of HSV immediate-early (IE) or delayed-early class promoters. DNA-transfected mouse Ltk+ cell lines harboring coselected IE175-IFN or thymidine kinase (TK)-IFN hybrid genes gave only low basal expression of human IFN. However, infection of both cell types with HSV type 1 or HSV type 2 produced abundant synthesis of IFN-specific RNA and biologically active IFN protein product. The IE175-IFN cell lines consistently gave 20- to 150-fold increases in IFN titers, and several TK-IFN cell lines yielded 100- to 500-fold induction. In the IE175-IFN cells, expression of IFN RNA also increased up to 200-fold and was detectable within 30 to 60 min after virus infection. Qualitatively similar results were obtained with hybrid G418-resistant Ltk- or Vero cell lines containing coselected IE175-CAT and TK-CAT constructs, except that there was relatively high basal expression of IE175-CAT. All three sets of IE cell lines (but not the delayed-early cell lines) responded to virus infection both in the presence of cycloheximide and with mutants defective in IE gene expression, demonstrating specific trans-activation by the pre-IE virion factor. In contrast, activation in the TK hybrid cell types required viral gene expression and the presence of a functional IE175 gene product. Up to 30-fold amplification in the copy number of the resident IFN or CAT DNA sequences also occurred within 20 h after HSV infection in IE175 hybrid cells but not in TK hybrid cells. Amplification was abolished either by treatment with phosphonacetate or by superinfection with a ts mutant unable to synthesize viral DNA, demonstrating specific HSV activation of the viral DNA replication origin (oriS) present in the IE hybrid constructs.

Acetyltransferases↗

PubMed2005

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins.

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

Animals↗

PubMed1988

Structure and organization of Marchantia polymorpha chloroplast genome. I. Cloning and gene identification.

We have determined the complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha, using a clone bank of chloroplast DNA fragments. The circular genome consists of 121,024 base-pairs and includes two large inverted repeats (IRA and IRB, each 10,058 base-pairs), a large single-copy region (LSC, 81,095 base-pairs), and a small single-copy region (SSC, 19,813 base-pairs). The nucleotide sequence was analysed with a computer to deduce the entire gene organization, assuming the universal genetic code and the presence of introns in the coding sequences. We detected 136 possible genes. 103 gene products of which are related to known stable RNA or protein molecules. Stable RNA genes for four species of ribosomal RNA and 32 species of tRNA were located, although one of the tRNA genes may be defective. Twenty genes encoding polypeptides involved in photosynthesis and electron transport were identified by comparison with known chloroplast genes. Twenty-five open reading frames (ORFs) show structural similarities to Escherichia coli RNA polymerase subunits, 19 ribosomal proteins and two related proteins. Seven ORFs are comparable with human mitochondrial NADH dehydrogenase genes. A computer-aided homology search predicted possible chloroplast homologues of bacterial proteins; two ORFs for bacterial 4Fe-4S-type ferredoxin, two for distinct subunits of a protein-dependent transport system, one ORF for a component of nitrogenase, and one for an antenna protein of a light-harvesting complex. The other 33 ORFs, consisting of 29 to 2136 codons, remain to be identified, but some of them seem to be conserved in evolution. Detailed information on gene identification is presented in the accompanying papers. We postulated that there were 22 introns in 20 genes (8 tRNA genes and 12 ORFs), which may be classified into the groups I and II found in fungal mitochondrial genes. The structural gene for ribosomal protein S12 is trans-split on the opposite DNA strand. The universal genetic code was confirmed by the substitution pattern of simultaneous codons, and by possible codon recognition of the chloroplast-encoded tRNA molecules, assuming no importation of tRNA molecules from the cytoplasm. The nucleotide residue A or T is preferred at the third position of the codons (G+C, 11.9%) and in intergenic spacers (G+C, 19.5%), resulting in an overall G+C content that is low (28.8%) throughout the liverwort chloroplast genome. Possible gene expression signals such as promoters and terminators for transcription, predicted locations of gene products, and DNA replicative origins are discussed.

Base Sequence↗

PubMed1998

Identification of a preinitiation step in DNA replication that is independent of origin recognition complex and cdc6, but dependent on cdk2.

Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and minichromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation complexes. Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA replication fails to initiate. We also find that after assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication. Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity.

Animals↗

PubMed1998

Silent chromatin in yeast: an orchestrated medley featuring Sir3p [corrected].

Extensive regions of chromosomes can be transcriptionally repressed through silencing mechanisms mediated by complex chromatin structures. One of the most refined molecular portraits of silenced chromatin comes from studies of the silent mating-type loci and telomeres of S. cerevisiae. In this budding yeast, the Sir3p silent information regulator emerges as a critically important silencing component that interacts with nucleosomes and other silencing proteins. Not only is it essential for silencing, but Sir3p is also capable of spreading silenced chromatin when its dosage is increased. Sir3p is a target of mitogen-activated protein (MAP) kinase cascade regulation and has significant similarity to the Orc1p subunit of the DNA replication origin recognition complex. Thus, in concert with other silencing proteins, Sir3p appears poised to respond to cellular signals and reprogram silencing through replication-associated assembly of repressive chromatin structures.

Chromatin↗

PubMed2006

Herpes simplex virus type 1 recombinants without the oriL sequence replicate DNA with increased fidelity.

Herpes simplex virus type 1 (HSV-1) contains three DNA replication origins (ori) of two types. The oriL is located in the center of the unique long sequences, whereas two copies of oriS, which are structurally different from oriL, are within the reiterated sequences flanking the unique short sequences. Recombinant viruses were constructed from ts+7, which contains a deletion of oriL sequences, to have either the beta-galactosidase gene or the supF amplicon integrated into the thymidine kinase locus. Rescue recombinants also were constructed from the supF-containing recombinant to restore the deleted oriL to the wild type sequences. These recombinants were subjected to mutagenesis assays. Results demonstrated that ts+7 viruses with the deletion in oriL sequences replicated both target genes with higher fidelity compared to those derived from the parental strain KOS. Possible mechanisms leading to the high fidelity of DNA replication mediated by viruses without intact oriL sequences are discussed.

Animals↗

PubMed2003

Expression of minichromosome maintenance proteins in vascular smooth muscle cells is ERK/MAPK dependent.

Proliferation of vascular smooth muscle cells (VSMC) represents a key event for the pathogenesis of postangioplasty restenosis. Minichromosome maintenance proteins (MCM) form essential components of the prereplicative complex at DNA replication origins and are regulated by E2F. The present studies were designed to investigate the signal transduction pathways controlling the expression of MCM6 and MCM7 in VSMC in response to mitogenic stimuli. MCM6 and MCM7 expression was substantially increased after stimulation with platelet-derived growth factor-BB and insulin. Pretreatment with PD98059, a specific inhibitor of the extracellular signal-regulated kinases (ERK)-mitogen-activated protein kinase (MAPK), competely inhibited the mitogen-induced MCM6 and MCM7 mRNA and protein expression, demonstrating a critical role for this pathway in transmitting transmembrane signals required for the initiation of DNA replication. The p38MAPK inhibitor SB203580, the phosphatidylinositol 3 kinase (PI3-kinase) pathway inhibitor wortmannin, and the protein kinase C pathway (PKC) inhibitor Gö 6976 did not significantly affect mitogen-induced MCM6 and MCM7 expression. Transient transfection experiments revealed that PD98059 inhibited mitogen-induced MCM6 and MCM7 transcriptional activation. In addition, blockade of ERK/MAPK signaling with PD98059 strongly inhibited phosphorylation of the retinoblastoma protein (Rb) and activity of a luciferase reporter plasmid driven by multiple E2F elements. Inhibition of mitogen-induced MCM6 and MCM7 expression by PD98059 was reversed by ectopic overexpression of E2F, indicating that ERK/MAPK signaling is required for events that occur upstream of E2F release from phosphorylated Rb. In combination, these data demonstrate that the ERK/MAPK signal transduction pathway plays a central role in regulating E2F-dependent MCM expression and DNA replication in VSMC.

Becaplermin↗

PubMed2005

ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA.

Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC-Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.

Amino Acid Sequence↗