Bending of the origin of replication of E. coli by binding of IHF at a specific site

In studies of DNA replication in Escherichia coli, an important question concerns the role of the initiator protein DnaA. This protein is known to bind to a specific 9-bp sequence in the origin of replication, but it is not understood how it can recognize another, relatively distant, 13-bp sequence that has no homology to the binding site but is where the DnaA protein serves its catalytic function in the initiation of DNA replication. This effect of DnaA might be achieved by bending of DNA in this region. I have searched for putative binding sites for integration host factor (IHF), a protein known to bend DNA. Here I report the finding of an IHF binding site in the E. coli origin and present direct evidence that IHF binds and causes DNA bending in this region. On the basis of these results I propose a model wherein formation of a higher-order nucleoprotein structure would facilitate the action of DnaA protein in the initiation events.     

Inheritance of the replication complex: a unique or common phenomenon in the control of DNA replication?

Early models of the regulation of initiation of DNA replication by protein complexes predicted that binding of a replication initiator protein to a replicator region is required for initiation of each DNA replication round, since after the initiation event the replication initiator should dissociate from DNA. It was, therefore, assumed that binding of the replication initiator is a signal for triggering DNA replication. However, more recent investigations have revealed that in many replicons this is not the case. Studies on the regulation of the replication of plasmids derived from bacteriophage lambda demonstrated that, once assembled, the replication complex can be inherited by one of the two daughter plasmid copies after each replication round and may function in subsequent replication rounds. Since this DNA-bound protein complex bears information about specific initiation of DNA replication, this phenomenon has been called "protein inheritance." A similar phenomenon has recently been reported for oriJ-based plasmids. Moreover, the current model of the initiation of DNA replication in the yeast Saccharomyces cerevisiae proposes that the origin recognition complex (ORC) remains bound to one copy of the ori sequence (the ARS region) after initiation of DNA replication. Thus, it seems plausible that protein inheritance is not unique for lambda plasmids, but may be a common phenomenon in the control of DNA replication, at least in microbes.     

Replication origins in metazoan chromosomes: fact or fiction?

The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition complex) next to an easily unwound DNA sequence at which replication can begin. The origin recognition complex provides a platform on which additional proteins assemble to form a pre-replication complex that can be activated at S-phase by specific protein kinases. Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication. Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial. In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone. Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification. Such complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development.     

Universal minicircle sequence-binding protein, a sequence-specific DNA-binding protein that recognizes the two replication origins of the kinetoplast DNA minicircle

Replication of the kinetoplast DNA minicircle lagging (heavy (H))-strand initiates at, or near, a unique hexameric sequence (5'-ACGCCC-3') that is conserved in the minicircles of trypanosomatid species. A protein from the trypanosomatid Crithidia fasciculata binds specifically a 14-mer sequence, consisting of the complementary strand hexamer and eight flanking nucleotides at the H-strand replication origin. This protein was identified as the previously described universal minicircle sequence (UMS)-binding protein (UMSBP) (Tzfati, Y., Abeliovich, H., Avrahami, D., and Shlomai, J. (1995) J. Biol. Chem. 270, 21339-21345). This CCHC-type zinc finger protein binds the single-stranded form of both the 12-mer (UMS) and 14-mer sequences, at the replication origins of the minicircle L-strand and H-strand, respectively. The attribution of the two different DNA binding activities to the same protein relies on their co-purification from C. fasciculata cell extracts and on the high affinity of recombinant UMSBP to the two origin-associated sequences. Both the conserved H-strand hexamer and its flanking nucleotides at the replication origin are required for binding. Neither the hexameric sequence per se nor this sequence flanked by different sequences could support the generation of specific nucleoprotein complexes. Stoichiometry analysis indicates that each UMSBP molecule binds either of the two origin-associated sequences in the nucleoprotein complex but not both simultaneously.     

The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout,Oncorhynchus mykiss

The complete nucleotide sequence of the mitochondrial DNA of the rainbow trout, Onchorynchus mykiss, has been determined. The total length of the molecule is 16,660 bp. The rainbow trout mitochondrial DNA has the same organization described in eutherian mammals, the clawed frog (Xenopus laevis), and the two fish species, Oriental stream loach (Crossotoma lacustre) and carp (Cyprinus carpio). Alignment and comparison of the deduced amino acid sequences of the 13 proteins encoded by rainbow trout and other vertebrate mitochondrial genomes allowed us to estimate that COI is the most conserved mitochondrial subunit (amino acid identity ranging from 85.6% to 94.8%) whereas ATPase 8 is the most variable one (amino acid identity ranging from 30.8% to 70.4%). Putative secondary structures for the 22 tRNAs found in the molecule are given along with an extensive comparison of tRNA sequences among representative species of each major group of vertebrates. In this sense, an unusual cloverleaf structure for the tRNA^Ser(AGY) is proposed. A stem-loop structure inferred for the origin of the L-strand replication (O_L) and the presence of a large polycytidine tract in the O_L loop is described. The existence of this stretch instead of the usual T-rich sequence reported so far in mammal mtDNAs is explained in terms of a less-strict template dependence of the RNA primase involved in the initiation of L-strand replication. Correspondence to: J.M. Bautista     

Sequence and structural requirements for primase initiation in the SV40 origin of replication.

Primase synthesizes decaribonucleotides for priming of lagging and possibly leading strand synthesis at a replication fork. The sites of initiation by purified mouse primase were shown to be highly specific within the SV40 origin of replication. This study further examines the role of the 27-bp inverted repeat in the origin for initiation. A site is observed on the L-strand template at nucleotide position (np) 22 positioned a similar distance from the 27-bp inverted repeat as sites previously reported on the E-strand. The initiations adjacent to the 27-bp repeat have a higher Km for rATP than other sites. A deletion within the inverted repeat eliminated initiation at sites proximal to the hairpin on both E and L strands but had no effect at more distant sites. A deletion mutant which left the inverted repeat intact but deleted the initiation sites at np 5210-5220 on the E-strand was not active as a template for proximal sites. These results indicate that primase has two modes of recognition, one that requires the SV40 inverted repeat structure and a specific sequence and another that requires sequence alone. Additional regions of the SV40 genome have also been examined and of approximately 2000 nucleotides of single stranded template examined, only one additional site was observed at np 2412 on the E-strand. This indicates that primase initiations are highly specific for the SV40 origin and their potential functional role is discussed.     

A binding protein (p82 protein) recognizes specifically the curved heterochromatic DNA in Artemia franciscana

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA replication, site specific recombination and DNA packaging. In Artemia franciscana (Phillopoda anostraca) cells we have revealed that an AluI DNA family of repeats, 113-bp in length, is the major component of the constitutive heterochromatin found in the species. By analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments and electrophoretic experiments we verified that the repetitive DNA shows a stable curvature that confers a solenoidal geometry to the double helix. Using the cloned monomeric fragment, as molecular probe, we describe the detection in an A. franciscana cell extract of a protein of 82 kDa (p82) that preferentially binds to heterochromatic DNA. This protein, purified of the other DNA binding proteins present in the crude cell extract, shows a greater affinity with the tandem copies of the AluI DNA fragment than with the monomer sequence. The binding of p82 protein to heterochromatic DNA is also drastically reduced in the presence of the antibiotic distamycin A, suggesting a role of the DNA curvature in the formation of the nucleoproteic complex.     

Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site

The replication origins (ORIs) of Schizosaccharomyces pombe, like those in most eukaryotes, are long chromosomal regions localized within A+T-rich domains. Although there is no consensus sequence, the interacting proteins are strongly conserved, suggesting that DNA structure is important for ORI function. We used atomic force microscopy in solution and DNA modelling to study the structural properties of the Spars1 origin. We show that this segment is the least stable of the surrounding DNA (9 kb), and contains regions of intrinsically bent elements (strongly curved and inherently supercoiled DNAs). The pORC-binding site co-maps with a superhelical DNA region, where the spatial arrangement of adenine/thymine stretches may provide the binding substrate. The replication initiation site (RIP) is located within a strongly curved DNA region. On pORC unwinding, this site shifts towards the apex of the curvature, thus potentiating DNA melting there. Our model is entirely consistent with the sequence variability, large size and A+T-richness of ORIs, and also accounts for the multistep nature of the initiation process, the specificity of pORC-binding site(s), and the specific location of RIP. We show that the particular DNA features and dynamic properties identified in Spars1 are present in other eukaryotic origins.     

Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase.

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.     

Molecular cloning of mtp70, a mitochondrial member of the hsp70 family.

We have isolated a gene from the protozoan parasite Trypanosoma cruzi that encodes a previously unidentified member of the 70-kilodalton heat shock protein (hsp70) family. Among all the eucaryotic hsp70 proteins described to date, this trypanosome protein, mtp70, is uniquely related in sequence and structure to the hsp70 of Escherichia coli, DnaK, which functions in the initiation of DNA replication. This relationship to DnaK is especially relevant in view of the intracellular location of the protein. Within the trypanosome, mtp70 is located in the mitochondrion, where it associates with kinetoplast DNA (kDNA), the unusual mitochondrial DNA that distinguishes this order of protozoa. Moreover, mtp70 is located in the specific region of the kinetoplast in which kDNA replication occurs. In view of the known functions of DnaK, the localization of mtp70 to the site of kDNA replication suggests that mtp70 may participate in eucaryotic mitochondrial DNA replication in a manner analogous to that of DnaK in E. coli.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

A novel protein binds a key origin sequence to block replication of an E. coli minichromosome

A sequence of three tandem repeats of a 13-mer in the replication origin (oriC) of E. coli is the highly conserved site of opening of the duplex for initiation of DNA synthesis. A protein that binds this sequence has been discovered in E. coli and purified to homogeneity. This novel 33 kd polypeptide behaves as a dimer. Binding to the 13-mers is specific and limited to this region. At a ratio of 10-20 monomers per oriC plasmid, the binding blocks initiation by preventing the opening of the 13-mer region by dnaA protein. Once the 13-mers are opened by dnaA protein action, the 33 kd protein is without effect on the subsequent stages of replication. The specificity of binding and profound inhibitory effect suggest a regulatory role for this protein at an early stage of chromosome initiation.     

Structure and function of the adenovirus origin of replication

Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (KD = 2 X 10(-11) M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation.     

Various elements of the structure of mini-ring kinetoplast DNA of Crithidia fasciculata

The nucleotide sequence of 1.4 kbp SmaI-fragment of minicircle DNA from kinetoplasts of Crithidia fasciculata has been determined and some sequence elements characterized. The sequence contains several oligo(dT)blocks located on the same strand in phase with a period of DNA helix turn, thus representing a "bent helix". Both sides of the bent helix region are flanked by sequences capable of forming a cloverleaf structure. There are also two direct 150 bp repeats located 180 degrees apart on the circular map of the molecule. Each repeat contains the sites of H-strand and L-strand replication origin. The specific stem-loop secondary structure may be folded by the nucleotide sequence within the origins region. The alignment of the sequence determined with two other C. fasciculata minicircle sequences spanning over the bent helix and the adjacent regions has indicated the presence of several conserved sequence blocks, one of them representing the sequence of the bend. The divergence of three sequences occurred mainly by small insertions-deletions. Several open reading frames were found, the largest of which being capable of coding for the approximately 200 amino acids polypeptide.