The E1 initiator recognizes multiple overlapping sites in the papillomavirus origin of DNA replication

A common feature of replicator sequences from a variety of organisms is multiple binding sites for an initiator protein. By binding to the replicator, initiators mark the site and contribute to melting or distortion of the DNA. We have defined the recognition sequence for the papillomavirus E1 initiator and determined the arrangement of binding sites in the viral origin of replication. We show that E1 recognizes a hexanucleotide sequence which is present in overlapping arrays in virtually all papillomavirus replicators. Binding of the initiator to these sites would result in the formation of a closely packed array of E1 molecules that wrap around the double helix.     

Two oppositely oriented arrays of low-affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre-RC assembly

The onset of chromosomal DNA replication requires highly precise and reproducible interactions between initiator proteins and replication origins to assemble a pre-replicative complex (pre-RC) that unwinds the DNA duplex. In bacteria, initiator protein DnaA, bound to specific high- and low-affinity recognition sites within the unique oriC locus, comprises the pre-RC, but how complex assembly is choreographed to ensure precise initiation timing during the cell cycle is not well understood. In this study, we present evidence that higher-order DnaA structures are formed at oriC when DnaA monomers are closely positioned on the same face of the DNA helix by interaction with two oppositely oriented essential arrays of closely spaced low-affinity DnaA binding sites. As DnaA levels increase, peripheral high-affinity anchor sites begin cooperative loading of the arrays, which is extended by sequential binding of additional DnaA monomers resulting in growth of the complexes towards the centre of oriC. We suggest that this polarized assembly of unique DnaA oligomers within oriC plays an important role in mediating pre-RC activity and may be a feature found in all bacterial replication origins.     

Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate.

PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication. MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome. However, this fork lacks the single-stranded DNA needed to load DnaB. Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA. The helicase activity of PriA could then open the duplex to create the DnaB binding site. In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template. PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction. Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro. This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication. Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks. As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication.     

Replicator dominance in a eukaryotic chromosome.

Replicators are genetic elements that control initiation at an origin of DNA replication (ori). They were first identified in the yeast Saccharomyces cerevisiae as autonomously replicating sequences (ARSs) that confer on a plasmid the ability to replicate in the S phase of the cell cycle. The DNA sequences required for ARS function on a plasmid have been defined, but because many sequences that participate in ARS activity are not components of chromosomal replicators, a mutational analysis of the ARS1 replicator located on chromosome IV of S. cerevisiae was performed. The results of this analysis indicate that four DNA elements (A, B1, B2 and B3) are either essential or important for ori activation in the chromosome. In a yeast strain containing two closely spaced and identical copies of the ARS1 replicator in the chromosome, only one is active. The mechanism of replicator repression requires the essential A element of the active replicator. This element is the binding site for the origin recognition complex (ORC), a putative initiator protein. The process that determines which replicator is used, however, depends entirely upon flanking DNA sequences.     

The human beta-globin replication initiation region consists of two modular independent replicators

Previous studies have shown that mammalian cells contain replicator sequences, which can determine where DNA replication initiates. However, the specific sequences that confer replicator activity were not identified. Here we report a detailed analysis of replicator sequences that dictate initiation of DNA replication from the human beta-globin locus. This analysis suggests that the beta-globin replication initiation region contains two adjacent, redundant replicators. Each replicator was capable of initiating DNA replication independently at ectopic sites. Within each of these two replicators, we identified short, discrete, nonredundant sequences, which cooperatively determine replicator activity. Experiments with somatic cell hybrids further demonstrated that the requirements for initiation at ectopic sites were similar to the requirements for initiation within native human chromosomes. The replicator clustering and redundancy exemplified in the human beta-globin locus may account for the extreme difficulty in identifying replicator sequences in mammalian cells and suggest that mammalian replication initiation sites may be determined by cooperative sequence modules.     

The replicator of the Epstein-Barr virus latent cycle origin of DNA replication, oriP, is composed of multiple functional elements

Replication of the Epstein-Barr virus genome initiates at one of several sites in latently infected, dividing cells. One of these replication origins is close to the viral DNA maintenance element, and, together, this replication origin and the maintenance element are referred to as oriP. The replicator of oriP contains four binding sites for Epstein-Barr virus nuclear antigen 1 (EBNA-1), the sole viral protein required for the replication and maintenance of oriP plasmids. We showed previously that these EBNA-1 sites function in pairs and that mutational inactivation of one pair does not eliminate replicator function. In this study we characterized the contribution of each EBNA-1 site within the replicator and flanking sequences through the use of an internally controlled replication assay. We present evidence that shows that all four EBNA-1 sites are required for an oriP plasmid to be replicated in every cell cycle. Results from these experiments also show that the paired EBNA-1 binding sites are not functionally equivalent and that the low affinity of sites 2 and 3 compared to that of sites 1 and 4 is not essential for replicator function. Our results suggest that a host cell protein(s) binds sequences flanking the EBNA-1 sites and that interactions between EBNA-1 and this protein(s) are critical for replicator function. Finally, we present evidence that shows that the minimal replicator of oriP consists of EBNA-1 sites 3 and 4 and two copies of a 14-bp repeat that is present in inverse orientation flanking these EBNA-1 sites. EBNA-1 sites 1 and 2, together with an element(s) within nucleotides 9138 to 9516, are ancillary elements required for full replicator activity.     

ATP-dependent minor groove recognition of TA base pairs is required for template melting by the E1 initiator protein

Template melting is an essential step in the initiation of DNA replication, but the mechanism of template melting is unknown for any replicon. Here we demonstrate that melting of the bovine papillomavirus type 1 ori is a sequence-dependent process which relies on specific recognition of TA base pairs in the minor groove by the E1 initiator. We show that correct template melting is a prerequisite for the formation of a stable double hexamer with helicase activity and that ori mutants that fail to melt correctly are defective for ori unwinding and DNA replication in vivo. Our results also indicate that melting of the DNA is achieved by destabilization of the double helix along its length through multiple interactions with E1, each of which is responsible for melting of a few base pairs, resulting in the extensive melting that is required for initiation of DNA replication.     

Identification and analysis of a lytic-phase origin of DNA replication in human herpesvirus 7.

Human herpesvirus 7 (HHV-7) DNA sequences colinear with the HHV-6 lytic-phase origin of DNA replication (oriLyt) were amplified by PCR. Plasmid constructs containing these sequences were replicated in HHV-7-infected cord blood mononuclear cells but not in HHV-6-infected cells. In contrast, plasmids bearing HHV-6 oriLyt were replicated in both HHV-6- and HHV-7-infected cells. Finally, the minimal HHV-7 DNA element necessary for replicator activity was mapped to a 600-bp region which contains two sites with high homology to the consensus binding site for the HHV-6 origin binding protein. At least one of these binding sites was shown to be essential for replicator function of HHV-7 oriLyt.     

Le modèle du réplicon est-il applicable aux eucaryotes supérieurs?

Thirty-five years ago, the Replicon model was proposed by Jacob, Brenner and Cuzin to explain the regulation of the Escherichia coli DNA replication. In this model, a genetic element, the replicator, would function as a target for a positive-acting initiator protein to drive the initiation of replication. This simple idea has been extremely useful in providing a framework to explain how the initiation of DNA replication occurs in all organisms. The identification of autonomously replicating sequences (ARSs) in budding yeast was the first extension of the Replicon model to eukaryotic chromosomes. In the higher eukaryotes, many biochemically defined replication start sites have been identified; nevertheless there is little genetic data indicating that these sites contain DNA sequences that are essential for replication. Moreover, in early Xenopus or Drosophila embryos, specific DNA sequences are not required either for initiating DNA replication or for preventing rereplication within a single cell cycle. This apparently fundamental difference between replicators in yeast and metazoan embryos may be more superficial than initially thought. In fact, during the past several years, an eukaryotic initiator conserved from yeast to man and also present in embryonic cells, the origin recognition complex (ORC), has been characterized, suggesting that the initiation mechanism should be essentially the same in prokaryotes and eukaryotes. In addition, the efficient once-per-cell-cycle replication of DNA is ensured in eukaryotes by a simple two-step mechanism in which the assembly of stable prereplicative complexes (PreRCs) at origins precedes and is temporally separated from the firing of these origins. Regulation of this process by cyclin-dependent kinases ensures that when origins fire, the cell is no longer competent to form new PreRCs. Now, it is important to understand how these complexes are remodeled or disassembled during replication initiation to trigger the transition from a stable origin-bound complex to a mobile replication machine.     

Building the bacterial orisome: high‐affinity DnaA recognition plays a role in setting the conformation of oriC DNA

During assembly of the E. coli pre‐replicative complex (pre‐RC), initiator DnaA oligomers are nucleated from three widely separated high‐affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low‐affinity DnaA recognition sites, but is also regulated by a switch‐like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively. Although their recognition sites are separated by up to 90 bp, Fis represses IHF binding and weak DnaA interactions until accumulating DnaA displaces Fis from oriC. It remains unclear whether high‐affinity DnaA binding plays any role in Fis repression at a distance and it is also not known whether all high‐affinity DnaA recognition sites play an equivalent role in oligomer formation. To examine these issues, we developed origin‐selective recombineering methods to mutate E. coli chromosomal oriC. We found that, although oligomers were assembled in the absence of any individual high‐affinity DnaA binding site, loss of DnaA binding at peripheral sites eliminated Fis repression, and made binding of both Fis and IHF essential. We propose a model in which interaction of DnaA molecules at high‐affinity sites regulates oriC DNA conformation.     

Differential binding of replication proteins across the human c-myc replicator

The binding of the prereplication complex proteins Orc1, Orc2, Mcm3, Mcm7, and Cdc6 and the novel DNA unwinding element (DUE) binding protein DUE-B to the endogenous human c-myc replicator was studied by chromatin immunoprecipitation. In G(1)-arrested HeLa cells, Mcm3, Mcm7, and DUE-B were prominent near the DUE, while Orc1 and Orc2 were least abundant near the DUE and more abundant at flanking sites. Cdc6 binding mirrored that of Orc2 in G(1)-arrested cells but decreased in asynchronous or M-phase cells. Similarly, the signals from Orc1, Mcm3, and Mcm7 were at background levels in cells arrested in M phase, whereas Orc2 retained the distribution seen in G(1)-phase cells. Previously shown to cause histone hyperacetylation and delocalization of replication initiation, trichostatin A treatment of cells led to a parallel qualitative change in the distribution of Mcm3, but not Orc2, across the c-myc replicator. Orc2, Mcm3, and DUE-B were also bound at an ectopic c-myc replicator, where deletion of sequences essential for origin activity was associated with the loss of DUE-B binding or the alteration of chromatin structure and loss of Mcm3 binding. These results show that proteins implicated in replication initiation are selectively and differentially bound across the c-myc replicator, dependent on discrete structural elements in DNA or chromatin.     

Bacterial initiators form dynamic filaments on single-stranded DNA monomer by monomer

DNA replication initiation is mediated across all domains of life by initiator proteins oligomerizing at replication origins. Recently, it was shown that initiators can directly bind single-stranded DNA (ssDNA) and thus might enhance origin melting. In this study, we used single-molecule fluorescence assays to probe the ssDNA binding mechanism of the replication initiator DnaA. Our experiments revealed that DnaA forms a dynamic filament on ssDNA in 3′ to 5′ directionality in the presence of ATP and analogs. After nucleation with a three-monomer seed, monomers dynamically assemble and disassemble one monomer at a time at the 5′ end, each monomer binding three nucleotides of ssDNA. The addition of adjacent double-stranded DnaA binding sites stabilized the DnaA filament on ssDNA. Our results extend the current models of origin melting via DnaA ssDNA interaction.     

Crystal structure of PriB, a component of the Escherichia coli replication restart primosome

Maintenance of genome stability following DNA damage requires origin-independent reinitiation of DNA replication at repaired replication forks. In E. coli, PriA, PriB, PriC, and DnaT play critical roles in recognizing repaired replication forks and reloading the replisome onto the template to reinitiate DNA replication. Here, we report the 2.0 A resolution crystal structure of E. coli PriB, revealing a dimer that consists of a single structural domain formed by two oligonucleotide/oligosaccharide binding (OB) folds. Structural similarity of PriB to single-stranded DNA binding proteins reveals insights into its mechanisms of DNA binding. The structure further establishes a putative protein interaction surface that may contribute to the role of PriB in primosome assembly by facilitating interactions with PriA and DnaT. This is the first high-resolution structure of a protein involved in oriC-independent replisome loading and provides unique insight into mechanisms of replication restart in E. coli.     

Adjacent residues in the E1 initiator beta-hairpin define different roles of the beta-hairpin in Ori melting, helicase loading, and helicase activity

We have analyzed two residues in the helicase domain of the E1 initiator protein. These residues are part of a highly conserved structural motif, the beta-hairpin, which is present in the helicase domain of all papovavirus initiator proteins. These proteins are unique in their ability to transition from local template melting activity to unwinding. We demonstrate that the beta-hairpin has two functions. First, it is the tool used by the E1 double trimer (DT) to pry open and melt double-stranded DNA. Second, it is required for the unwinding activity of the hexameric E1 helicase. The fact that the same structural element, but not the same residues, contacts both dsDNA in the DT for melting and ssDNA in the double hexamer (DH) for helicase activity provides a link between local origin melting and DNA helicase activity and suggests how the transition between these two states comes about.     

Novel second-site suppression of a cold-sensitive defect in phage P22 procapsid assembly

The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalton gene 1 protein. The assembly of this dodecameric complex at a unique capsid vertex requires scaffolding subunits. The mechanism that ensures the location of the 12-fold symmetrical portal at only one of the 12 5-fold vertices of an icosahedral virus capsid presents a unique assembly problem, which, in some viruses, is solved by the portal also acting as initiator of procapsid assembly. Phage P22 procapsids, however, are formed in the absence of the portal protein. The 1-csH137 mutation prevents the incorporation of the portal protein into procapsids. In a mixed infection with cs+ phage, the mutant subunits are able to form functional portals, suggesting that the cold-sensitivity does not affect portal-portal interactions, but affects the interaction of portal subunits with some other molecular species involved in the initiation of portal assembly. Interestingly, the cs defect is suppressed by temperature-sensitive folding mutations at four sites in the P22 tailspike gene 9. The suppression is allele-specific; other tailspike tsf mutations fail to suppress the cs defect. Translation through a suppressor site is required for suppression. This observation is unexpected, since analysis of nonsense mutations in this gene indicates that it is not required for procapsid assembly. Examination of the nucleic acid sequences in the neighborhood of each of the suppressor sites shows significant sequence similarity with the scaffolding gene translational initiation region on the late message. This supports a previously proposed model, in which procapsid assembly is normally initiated in a region on the late messenger RNA that includes the gene 8 start site. By this model, the suppressor mutations may be acting through protein-RNA interactions, changing sequences that identify alternative or competing sites at which the mutant portal subunits may be organized for assembly into the differentiated vertex of the phage capsid.     

Structure-based prediction of DNA target sites by regulatory proteins

Regulatory proteins play a critical role in controlling complex spatial and temporal patterns of gene expression in higher organism, by recognizing multiple DNA sequences and regulating multiple target genes. Increasing amounts of structural data on the protein-DNA complex provides clues for the mechanism of target recognition by regulatory proteins. The analyses of the propensities of base-amino acid interactions observed in those structural data show that there is no one-to-one correspondence in the interaction, but clear preferences exist. On the other hand, the analysis of spatial distribution of amino acids around bases shows that even those amino acids with strong base preference such as Arg with G are distributed in a wide space around bases. Thus, amino acids with many different geometries can form a similar type of interaction with bases. The redundancy and structural flexibility in the interaction suggest that there are no simple rules in the sequence recognition, and its prediction is not straightforward. However, the spatial distributions of amino acids around bases indicate a possibility that the structural data can be used to derive empirical interaction potentials between amino acids and bases. Such information extracted from structural databases has been successfully used to predict amino acid sequences that fold into particular protein structures. We surmised that the structures of protein-DNA complexes could be used to predict DNA target sites for regulatory proteins, because determining DNA sequences that bind to a particular protein structure should be similar to finding amino acid sequences that fold into a particular structure. Here we demonstrate that the structural data can be used to predict DNA target sequences for regulatory proteins. Pairwise potentials that determine the interaction between bases and amino acids were empirically derived from the structural data. These potentials were then used to examine the compatibility between DNA sequences and the protein-DNA complex structure in a combinatorial "threading" procedure. We applied this strategy to the structures of protein-DNA complexes to predict DNA binding sites recognized by regulatory proteins. To test the applicability of this method in target-site prediction, we examined the effects of cognate and noncognate binding, cooperative binding, and DNA deformation on the binding specificity, and predicted binding sites in real promoters and compared with experimental data. These results show that target binding sites for several regulatory proteins are successfully predicted, and our data suggest that this method can serve as a powerful tool for predicting multiple target sites and target genes for regulatory proteins.