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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↗
PubMed1997

Initiation mechanisms in replication of filamentous phage DNA.

Filamentous phage DNA replication occurs in two steps. First, an RNA-primed minus strand is made on the plus strand. Then, a new plus strand is made on the resulting double strand. The RNA primer which initiates synthesis of the minus strand is produced at a specific site on the plus strand template by the host RNA polymerase holoenzyme. Despite a lack of sequences similar to the promoter consensus, the DNA replication origin has a much higher affinity for the holoenzyme than the transcriptional promoters. The non-template strand of the single-stranded -10 region of the origin appears to be responsible for this high affinity. The recognition mechanisms seem to share common features with those in transcriptional promoters. Plus-strand synthesis is initiated by a specific nick introduced into the negatively supercoiled replicative form by the phage-encoded initiator protein. The nicking reaction is preceded by an ordered series of protein-induced DNA conformational changes.

Bacteriophages↗
PubMed2002

Drf1, a novel regulatory subunit for human Cdc7 kinase.

Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs. Cdc7, an evolutionarily conserved serine-threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins. Binding of the cell cycle-regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity. This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells. Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4. Drf1 is a nuclear cell cycle-regulated protein, it binds to Cdc7 and activates the kinase. Therefore, human Cdc7, like cyclin-dependent kinases, can be activated by alternative regulatory subunits. Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Statistical bioinformatic methods in microbial genome analysis.

It is probable that, increasingly, genome investigations are going to be based on statistical formalization. This review summarizes the state of art and potentiality of using statistics in microbial genome analysis. First, I focus on recent advances in functional genomics, such as finding genes and operons, identifying gene conversion events, detecting DNA replication origins and analysing regulatory sites. Then I describe how to use phylogenetic methods in genome analysis and methods for genome-wide scanning for positively selected amino acids. I conclude with speculations on the future course of genome statistical modeling.

Computational Biology↗
PubMed1994

A versatile plasmid vector system for the regulated expression of genes in Escherichia coli.

A series of plasmid expression vectors, which support the regulated and efficient expression of genes in Escherichia coli, have been constructed. The vectors consist of a DNA replication origin cassette, a promoter cassette, an efficient ribosome binding site together with a polylinker region and a lacZ gene. Several types of replication origins and promoter sequences are each available on cassettes. Fusion of the 5' TG dinucleotide of the gene under consideration to the A nucleotide, present on the vector, results in an ATG start codon and allows, in combination with the plasmid-borne ribosome binding site, the efficient expression of the cloned gene. Additionally, a second fusion of the gene at its 3' end with the lacZ gene, which is available in all three reading frames relative to the polylinker region, allows rapid selection of the correctly fused genes. As an example of the cloning of a regulatory gene, this vector system was used for the expression of the dnaA gene, of Escherichia coli, the initiator protein for DNA replication.

Bacterial Proteins↗
PubMed2003

Cyclin E ablation in the mouse.

E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.

Animals↗
PubMed2000

Polymorphism and divergence in the beta-globin replication origin initiation region.

DNA sequence polymorphism and divergence was examined in the vicinity of the human beta-globin gene cluster origin of replication initiation region (IR), a 1.3-kb genomic region located immediately 5' of the adult-expressed beta-globin gene. DNA sequence variation in the replication origin IR and 5 kb of flanking DNA was surveyed in samples drawn from two populations, one African (from the Gambia, West Africa) and the other European (from Oxford, England). In these samples, levels of nucleotide and length polymorphism in the IR were found to be more than two times as high as adjacent non-IR-associated regions (estimates of per-nucleotide heterozygosity were 0.30% and 0.12%, respectively). Most polymorphic positions identified in the origin IR fall within or just adjacent to a 52-bp alternating purine-pyrimidine ((RY)n) sequence repeat. Within- and between-populations divergence is highest in this portion of the IR, and interspecific divergence in the same region, determined by comparison with an orthologous sequence from the chimpanzee, is also pronounced. Higher levels of diversity in this subregion are not, however, primarily attributable to slippage-mediated repeat unit changes, as nucleotide substitution contributes disproportionately to allelic heterogeneity. An estimate of helical stability in the sequenced region suggests that the hypervariable (RY)n constitutes the major DNA unwinding element (DUE) of the replication origin IR, the location at which the DNA duplex first unwinds and new strand synthesis begins. These findings suggest that the beta-globin IR experiences a higher underlying rate of neutral mutation than do adjacent genomic regions and that enzyme fidelity associated with the initiation of DNA replication at this origin may be compromised. The significance of these findings for our understanding of eukaryotic replication origin biology is discussed.

Animals↗
PubMed1997

Initiation of DNA replication in eukaryotic cells.

The recent identification of proteins that recognize origins of DNA replication and control the initiation of eukaryotic DNA replication has provided critical molecular tools to dissect this process. Dynamic changes in the assembly and disassembly of protein complexes at origins are important for the initiation of DNA replication and occur throughout the cell cycle. Herein, we review the key proteins required for the initiation of DNA replication, their involvement in the protein complex assembly at replication origins, and how the cell cycle machinery regulates this process.

Animals↗
PubMed2002

A murine model of Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, and predisposition to hematopoietic malignancy. The clinical and cellular phenotypes of NBS substantially overlap those of ataxia-telangiectasia (A-T). NBS is caused by mutation of the NBS1 gene, which encodes a member of the Mre11 complex, a trimeric protein complex also containing Mre11 and Rad50. Several lines of evidence indicate that the ataxia-telangiectasia mutated (ATM) kinase and the Mre11 complex functionally interact. Both NBS and A-T cells exhibit ionizing radiation (IR) sensitivity and defects in the intra S phase checkpoint, resulting in radioresistant DNA synthesis (RDS)-the failure to suppress DNA replication origin firing after IR exposure. NBS1 is phosphorylated by ATM in response to IR, and this event is required for activation of the intra S phase checkpoint (the RDS checkpoint). We derived a murine model of NBS, the Nbs1(DeltaB/DeltaB) mouse. Nbs1(DeltaB/DeltaB) cells are phenotypically identical to those established from NBS patients. The Nbs1(DeltaB) allele was synthetically lethal with ATM deficiency. We propose that the ATM-Mre11 complex DNA damage response pathway is essential and that ATM or the Mre11 complex serves as a nexus to additional components of the pathway.

Amino Acid Sequence↗
PubMed1981

Nucleotide sequence of the asnA gene coding for asparagine synthetase of E. coli K-12.

We have subcloned the asnA gene of E. coli K-12, a gene coding for asparagine synthetase, from a previously cloned 6 mega-dalton segment of E. coli chromosome containing the DNA replication origin, ori, and asnA. The complete nucleotide sequence of the asnA gene was determined: the region of the structural gene extends 990 base-pairs at nucleotide positions 1434-2423 (see Fig. 3), which codes for a polypeptide of 330 amino-acid residues with a molecular weight of 36,688 daltons. The nucleotide sequences of the promoter and the ribosome-binding site of the gene are also assigned. We discuss the properties of its polypeptide.

Amino Acid Sequence↗
PubMed1995

Genetic analysis of the minimal replicon of plasmid pIP417 and comparison with the other encoding 5-nitroimidazole resistance plasmids from Bacteroides spp.

The nucleotide sequence of the DNA replication origin region of a Bacteroides vulgatus plasmid, pIP417, encoding 5-nitroimidazole resistance has been determined. This region of 1934 bp presents some characteristics similar to those of other replication protein-dependent origins. It contains a large open reading frame which could encode a basic Rep protein (RepA) of 36.8 kDa. Upstream of this ORF exist an AT-rich region, three direct repeats (iterons) of 21 bp, multiple DnaA binding sites, and sites, and sites for the integration host factor (IHF). Moreover, the amino acid sequence of the pIP417 RepA protein shows similarities with those of other Rep proteins encoded by plasmids of gram-negative bacteria: pRO1600 from Pseudomonas aeruginosa; pPS10 from Pseudomonas syringae; pFA3 from Neisseria gonorrhoeae; and two cryptic plasmids from Campylobacter hyointestinalis and Butyrivibrio fibrisolvens. Although RepA can be expressed in an Escherichia coli in vitro transcription-translation assay, vectors containing the pIP417 replication origin did not replicate in E. coli. The homology of the pIP417 replication region with the corresponding regions of other Bacteroides spp, plasmids was also studied by Southern blot hybridization. The results indicated that the repA gene of plasmid pIP417 is homologous to that of plasmid pIP421, but not of plasmid pIP419. The replication region of plasmid pIP421 was sequenced and showed about 80% identity at the nucleotide level with that of pIP417. A small (3634-bp) cloning vector (pFK12) of entirely defined nucleotide sequence was constructed for Bacteroides spp.

Amino Acid Sequence↗
PubMed1997

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts.

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin (oriA). Transformants were recovered only with the plasmid containing oriA, and all transformants contained an integrated plasmid copy at oriA, suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.

Animals↗
PubMed1993

Roles of the G site and phi X174-type primosome assembly site in priming of leading-strand synthesis: initiation by a mobile primosome and replication-fork arrest by RepA protein bound to oriR.

Bacterial replicons often contain single-strand initiation sequences (ssi) such as a G site (a sequence recognized by a dnaG-encoded primase for the synthesis of primer RNA) and a primosome assembly site (pas) near the DNA replication origin (ori). The R1 plasmid contains a G site downstream from oriR, which serves for the priming of the leading-strand synthesis of this plasmid. On the other hand, the F, R6K and Rts1 plasmids carry pas at similar locations relative to the respective ori. In order to assess the functional significance of these pas, R1 plasmid derivatives carrying an n'-pas (phi X174-type pas) in place of the G site were constructed and their replication properties were examined in vitro. Deletion of the G site in the R1 plasmid resulted in a nearly 80% reduction of total DNA synthesis in vitro, which was recovered to the wild-type (wt) level by inserting the G4 complementary ori. Furthermore, insertion of an n'-pas on the leading-strand template restored the in vitro replicative activity to a level 70% of wt. This recovery was dependent on the assembly of the phi X174-type primosome, which efficiently primed leading-strand synthesis and moved toward the oriR. However, the R1 plasmid derivative containing the n'-pas replicated unidirectionally in vitro, probably due to the anti-helicase activity of the RepA protein bound to oriR, which was shown by helicase assays using partial heteroduplexes as substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1988

Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation.

Functional upstream activator sequences (TAATGARAT motifs) of herpes simplex virus immediate-early genes were identified and shown both to bind a factor (TRF) present in uninfected HeLa cells and to confer inducibility by the virus regulatory protein, Vmw65, on a normally nonresponsive promoter. Point-mutation analyses demonstrated binding specificity and correlated binding with Vmw65 induction. Furthermore, the octamer domains of the adenovirus DNA replication origin, the histone H2B, and the immunoglobulin light chain genes bound and competed for TRF. The immunoglobulin octamer also conferred Vmw65 inducibility on the TK promoter. In addition, a modified form of TRF was specifically detected in infected cells. We conclude that TRF is similar or identical to the previously described octamer binding protein and is likely to be the target for coordinate induction of immediate-early gene expression by Vmw65.

Base Sequence↗
PubMed1980

Isolation of a replication origin complex from Escherichia coli.

A complex consisting of replicative origin DNA and several proteins was isolated from Escherichia coli. Cells of temperature-sensitive mutants were labeled at the origin and fractionated by sucrose gradient centrifugation. A complex highly purified in origin DNA sedimented as a unique band. This complex dissociated at high concentration, above 0.2 M KCl. Upon dialysis, the complex reformed, allowing further purification of its constituents. Three major protein bands were found, corresponding to proteins of the outer membrane. The complex did not sediment with membrane fractions, but adhered to the outer membrane in the presence of magnesium.

Bacterial Proteins↗
PubMed1986

Evolutionary changes of transcriptional control region in a minute-plaque viable deletion mutant of BK virus.

Two plaque morphology BK virus (BKV) mutants (pm526 and pm527) rescued from a hamster pineocytoma cell line Pc13 were characterized and compared with a similarly rescued and previously characterized mutant (pm522), its derivatives (tr530, tr531, and tr532), and the wild type (501) for their biological activities and for the structures of their transcriptional control regions. The two mutants grew somewhat more slowly in human embryonic kidney cells but transformed rat 3Y1 cells more efficiently than did the wild-type BKV. BKV pm526 formed minute plaques and had the shortest transcriptional control region, having only one 68-base-pair element, which is triplicated in the wild-type BKV. BKV pm526 was unstable during repeated replication in human embryonic kidney cells and yielded large-plaque viruses with longer HindIII C segments encompassing the BKV DNA replication origin. Comparison of nucleotide sequences of the transcriptional control regions among the mutants and the wild type showed that pm526 is a parent virus, from which all the other mutants had evolved, and that the evolutionary changes of the plaque size, from minute to small to large, were due to duplications of a certain segment containing the adenovirus E1A enhancer core or the simian virus 40 enhancer core found in the BKV 68-base-pair element. The activities to enhance early transcription, as measured by the ability to direct the synthesis of chloramphenicol acetyltransferase, approximately paralleled the plaque size. The duplication containing the adenovirus E1A enhancer core did not affect the transforming capacity of the parent virus, but the duplication including the simian virus 40 enhancer core significantly lowered the transforming capacity for rat cells.

Animals↗
PubMed1999

Identification of the Marek's disease virus serotype 2 genes homologous to the glycoprotein B (UL27), ICP18.5 (UL28) and major DNA-binding protein (UL29) genes of herpes simplex virus type 1.

We determined the nucleotide sequence of non-pathogenic Marek's disease virus serotype 2 (MDV2) strain HPRS24 glycoprotein B (gB) (UL27), ICP18.5 (UL28) and major DNA-binding protein (MDBP) (UL29) genes homologous to herpes simplex virus type 1 (HSV-1). The sequence data revealed that important motives in the proteins are conserved in MDV2 ICP18.5 and MDBP, however the sequence of viral DNA replication origin which exists in the regions between the UL29 and UL30 genes of other alphaherpesviruses was not found in the regions of the MDV2 genome. By northern blot analyses, we also demonstrated that 8.9, 5.0 and 2.6 kb transcripts were actually transcribed from the sequenced region in MDV2-infected cells. The MDV2 UL28 and UL29 genes have not been reported in other serotypes of MDV.

Amino Acid Sequence↗
PubMed1985

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Site and direction of initial DNA synthesis.

In vivo, T7 DNA replication is initiated 15% of the distance from the genetic left end of the chromosome. This site, the primary origin of replication, consists of a 200-base pair (bp) intergenic segment from 14.5 to 15.0% within which are located two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by a 61-bp AT-rich (79% A + T) region. A fragment of T7 DNA containing the primary origin has been inserted into plasmids in order to facilitate studies on initiation in vitro. Initiation of DNA synthesis can be reconstituted using T7 RNA polymerase, T7 DNA polymerase, and T7 origin-containing plasmid DNAs. DNA synthesis is stimulated greatly by the T7 gene 4 protein, an enzyme that has helicase and primase activities. When T7 gene 4 protein is present, replication primarily yields partially replicated Y-form molecules as observed by electron microscopy. Synthesis is unidirectional and the branches of the Y-form molecules are uniform in size, with the branch point of the Y located at the origin. Using restriction enzyme analysis, DNA synthesis has been shown to proceed in the same direction (rightward with respect to the T7 genetic map) as transcription from the two promoters located at the origin. Initiation of DNA synthesis in the opposite direction requires the addition of a single-stranded DNA-binding protein (Fuller, C.W., and Richardson, C.C. (1985) J. Biol. Chem. 260, 3197-3206). The initial products of DNA synthesis have been analyzed by polyacrylamide gel electrophoresis. These DNAs have 10 to 60 ribonucleotides covalently linked to their 5' termini. These RNA primers arise by transcription from each of the two promoters, phi 1.1A and phi 1.1B, located within the primary origin.

Base Sequence↗