A common mechanism for the ATP-DnaA-dependent formation of open complexes at the replication origin

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.     

Structural and thermodynamic signatures of DNA recognition by Mycobacterium tuberculosis DnaA

An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 Å resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 Å). These structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.     

Interaction of initiator proteins with the origin of replication of an IncL/M plasmid

The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.     

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.     

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.     

Highly organized DnaA-oriC complexes recruit the single-stranded DNA for replication initiation

In Escherichia coli, the replication origin oriC consists of two functional regions: the duplex unwinding element (DUE) and its flanking DnaA-assembly region (DAR). ATP-DnaA molecules multimerize on DAR, unwinding DUE for DnaB helicase loading. However, DUE-unwinding mechanisms and functional structures in DnaA-oriC complexes supporting those remain unclear. Here, using various in vitro reconstituted systems, we identify functionally distinct DnaA sub-complexes formed on DAR and reveal novel mechanisms in DUE unwinding. The DUE-flanking left-half DAR carrying high-affinity DnaA box R1 and the ATP-DnaA-preferential DnaA box R5, τ1-2 and I1-2 sites formed a DnaA sub-complex competent in DUE unwinding and ssDUE binding, thereby supporting basal DnaB loading activity. This sub-complex is further subdivided into two; the DUE-distal DnaA sub-complex formed on the ATP-DnaA-preferential sites binds ssDUE. Notably, the DUE-flanking, DnaA box R1-DnaA sub-complex recruits DUE to the DUE-distal DnaA sub-complex in concert with a DNA-bending nucleoid protein IHF, thereby promoting DUE unwinding and binding of ssDUE. The right-half DAR-DnaA sub-complex stimulated DnaB loading, consistent with in vivo analyses. Similar features are seen in DUE unwinding of the hyperthermophile, Thermotoga maritima, indicating evolutional conservation of those mechanisms.     

A critical DnaA box directs the cooperative binding of the Escherichia coli DnaA protein to the plasmid RK2 replication origin.

The requirement of DnaA protein binding for plasmid RK2 replication initiation the Escherichia coli was investigated by constructing mutations in the plasmid replication origin that scrambled or deleted each of the four upstream DnaA boxes. Altered origins were analyzed for replication activity in vivo and in vitro and for binding to the E. coli DnaA protein using a gel mobility shift assay and DNase I footprinting. Most strikingly, a mutation in one of the boxes, box 4, abolished replication activity and eliminated stable DnaA protein binding to all four boxes. Unlike DnaA binding to the E. coli origin, oriC, DnaA binding to two of the boxes (boxes 4 and 3) in the RK2 origin, oriV, is cooperative with box 4 acting as the "organizer" for the formation of the DnaA-oriV nucleoprotein complex. Interestingly, the inversion of box 4 also abolished replication activity, but did not result in a loss of binding to the other boxes. However, DnaA binding to this mutant origin was no longer cooperative. These results demonstrate that the sequence, position, and orientation of box 4 are crucial for cooperative DnaA binding and the formation of a nucleoprotein structure that is functional for the initiation of replication.     

Drunken-cell footprints: nuclease treatment of ethanol-permeabilized bacteria reveals an initiation-like nucleoprotein complex in stationary phase replication origins.

The nucleoprotein complex formed on oriC, the Escherichia coli replication origin, is dynamic. During the cell cycle, high levels of the initiator DnaA and a bending protein, IHF, bind to oriC at the time of initiation of DNA replication, while binding of Fis, another bending protein, is reduced. In order to probe the structure of nucleoprotein complexes at oriC in more detail, we have developed an in situ footprinting method, termed drunken-cell footprinting, that allows enzymatic DNA modifying reagents access to intracellular nucleoprotein complexes in E.coli, after a brief exposure to ethanol. With this method, we observed in situ binding of Fis to oriC in exponentially growing cells, and binding of IHF to oriC in stationary cells, using DNase I and Bst NI endonuclease, respectively. Increased binding of DnaA to oriC in stationary phase was also noted. Because binding of DnaA and IHF results in unwinding of oriC in vitro, P1 endonuclease was used to probe for intracellular unwinding of oriC. P1 cleavage sites, localized within the 13mer unwinding region of oriC ', were dramatically enhanced in stationary phase on wild-type origins, but not on mutant versions of oriC unable to unwind. These observations suggest that most oriC copies become unwound during stationary phase, forming an initiation-like nucleoprotein complex.     

Stoichiometry of DnaA and DnaB protein in initiation at the Escherichia coli chromosomal origin

Initiation of DNA replication at the Escherichia coli chromosomal origin, oriC, occurs through an ordered series of events that depend first on the binding of DnaA protein, the replication initiator, to DnaA box sequences within oriC followed by unwinding of an AT-rich region near the left border. The prepriming complex then forms, involving the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We assembled and isolated the prepriming complexes on an oriC plasmid, then determined the stoichiometries of proteins in these complexes by quantitative immunoblot analysis. DnaA protein alone binds to oriC with a stoichiometry of 4-5 monomers per oriC DNA. In the prepriming complex, the stoichiometries are 10 DnaA monomers and 2 DnaB hexamers per oriC plasmid. That only two DnaB hexamers are bound, one for each replication fork, suggests that the binding of additional molecules of DnaA in forming the prepriming complex restricts the loading of additional DnaB hexamers that can bind at oriC.     

DNA binding specificity of the replication initiator protein, DnaA from Helicobacter pylori

The key protein in the initiation of Helicobacter pylori chromosome replication, DnaA, has been characterized. The amount of the DnaA protein was estimated to be approximately 3000 molecules per single cell; a large part of the protein was found in the inner membrane. The H.pylori DnaA protein has been analysed using in vitro (gel retardation assay and surface plasmon resonance (SPR)) as well as in silico (comparative computer modeling) studies. DnaA binds a single DnaA box as a monomer, while binding to the fragment containing several DnaA box motifs, the oriC region, leads to the formation of high molecular mass nucleoprotein complexes. In comparison with the Escherichia coli DnaA, the H.pylori DnaA protein exhibits lower DNA-binding specificity; however, it prefers oriC over non-box DNA fragments. As determined by gel retardation techniques, the H.pylori DnaA binds with a moderate level of affinity to its origin of replication (4nM). Comparative computer modelling showed that there are nine residues within the binding domain which are possible determinants of the reduced H.pylori DnaA specificity. Of these, the most interesting is probably the triad PTL; all three residues show significant divergence from the consensus, and Thr398 is the most divergent residue of all.     

Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin.

Initiation of DNA synthesis is triggered by the binding of proteins to replication origins. However, little is known about the order in which specific proteins associate with origin sites during the cell cycle. We show that in cycling cells there are at least two different nucleoprotein complexes at oriC. A factor for inversion stimulation (FIS)-bound nucleoprotein complex, present throughout the majority of the cell cycle, switches to an integration host factor (IHF)-bound form as cells initiate DNA replication. Coincident with binding of IHF, initiator DnaA binds to its previously unoccupied R3 site. In stationary phase, a third nucleoprotein complex forms. FIS is absent and inactive oriC forms a nucleoprotein structure containing IHF that is not observed in cycling cells. We propose that interplay between FIS and IHF aids assembly of initiation nucleoprotein complexes during the cell cycle and blocks initiation at inappropriate times. This exchange of components at replication origins is reminiscent of switching between pre- and post-replicative chromatin states at yeast ARS1.     

The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces

Integration into a particular location in human chromosomes is a unique property of the adeno-associated virus (AAV). This reaction requires the viral Rep protein and AAV origin sequences. To understand how Rep recognizes DNA, we have determined the structures of the Rep endonuclease domain separately complexed with two DNA substrates: the Rep binding site within the viral inverted terminal repeat and one of the terminal hairpin arms. At the Rep binding site, five Rep monomers bind five tetranucleotide direct repeats; each repeat is recognized by two Rep monomers from opposing faces of the DNA. Stem-loop binding involves a protein interface on the opposite side of the molecule from the active site where ssDNA is cleaved. Rep therefore has three distinct binding sites within its endonuclease domain for its different DNA substrates. Use of these different interfaces generates the structural asymmetry necessary to regulate later events in viral replication and integration.     

Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus

Archaeal cell division cycle protein 6 (Cdc6)/Origin Replication Complex subunit 1 (Orc1) proteins share sequence homology with eukaryotic DNA replication initiation factors but are also structurally similar to the bacterial initiator DnaA. To better understand whether Cdc6/Orc1 functions in an eukaryotic or bacterial-like manner, we have characterized the interaction of two Cdc6/Orc1 paralogs (mthCdc6-1 and mthCdc6-2) with the replication origin from Methanothermobacter thermoautotrophicus. We show that while both proteins display a low affinity for a small dsDNA of random sequence, mthCdc6-1 binds tightly to a short duplex containing a single copy of a 13 bp sequence that is repeated throughout the origin. Surprisingly, sequence comparisons show that this 13 bp sequence is a minimized version of the Origin Recognition Box element found in many euryarchaeotal origins. Analysis of mthCdc6-1 mutants demonstrates that the helix–turn–helix motif in the winged-helix domain mediates the interaction with this sequence. Association of both mthCdc6/Orc1 paralogs with the duplex containing the minimized Origin Recognition Box fits to an independent binding sites model, but their interaction with longer DNA ligands is cooperative. Together, our data provide the first detailed biophysical characterization of the association of an archaeal DNA replication initiator with its origin. Our observations also indicate that the origin-binding properties of Cdc6/Orc1 proteins closely resemble those of bacterial DnaA.     

Two enhancer elements for DNA replication of pSC101, par and a palindromic binding sequence of the Rep protein.

The minimal replication origin (ori) of the plasmid pSC101 has been previously defined as an approximately 220-bp region by using plasmids defective in the par region, which is a cis-acting determinant of plasmid stability. This ori region contains the DnaA binding sequence, three repeated sequences (iterons), and an inverted repeat (IR) element (IR-1), one of the binding sites of an initiator protein, Rep (or RepA). In the present study, we show that plasmids containing par can replicate at a nearly normal copy number in the absence of IR-1 but still require a region (the downstream region) between the third iteron and IR-1. Because par is dispensable in plasmids retaining IR-1, par and IR-1 can compensate each other for efficient replication. The region from the DnaA box to the downstream region can support DNA replication at a reduced frequency, and it is designated "core-ori." Addition of either IR-1 or par to core-ori increases the copy number of the plasmid up to a nearly normal level. However, the IR-1 element must be located downstream of the third iteron (or upstream of the rep gene) to enhance replication of the plasmid, while the par region, to which DNA gyrase can bind, functions optimally regardless of its location. Furthermore, the enhancer activity of IR-1 is dependent on the helical phase of the DNA double helix, suggesting that the Rep protein bound to IR-1 stimulates the activation of ori via its interaction with another factor or factors capable of binding to individual loci within ori.     

Anatomy of the replication origin of plasmid ColE2-P9

The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.     

DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.     

Quantitative analysis of the mechanism of DNA binding by Bacillus DnaA protein

DnaA protein has the sole responsibility of initiating a new round of DNA replication in prokaryotic organisms. It recognizes the origin of DNA replication, and initiates chromosomal DNA replication in the bacterial genome. In Gram-negative Escherichia coli, a large number of DnaA molecules bind to specific DNA sequences (known as DnaA boxes) in the origin of DNA replication, oriC, leading to the activation of the origin. We have cloned, expressed, and purified full-length DnaA protein in large quantity from Gram-positive pathogen Bacillus anthracis (DnaA_BA). DnaA_BA was a highly soluble monomeric protein making it amenable to quantitative analysis of its origin recognition mechanisms. DnaA_BA bound DnaA boxes with widely divergent affinities in sequence and ATP-dependent manner. In the presence of ATP, the K_D ranged from 3.8 × 10⁻⁸ M for a specific DnaA box sequence to 4.1 × 10⁻⁷ M for a non-specific DNA sequence and decreased significantly in the presence of ADP. Thermodynamic analyses of temperature and salt dependence of DNA binding indicated that hydrophobic (entropic) and ionic bonds contributed to the DnaA_BA·DNA complex formation. DnaA_BA had a DNA-dependent ATPase activity. DNA sequences acted as positive effectors and modulated the rate (V_max) of ATP hydrolysis without any significant change in ATP binding affinity.     

Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface

Human replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), eukaryotic single-stranded DNA (ssDNA) binding protein required for DNA recombination, repair, and replication. The three subunits of human RPA are composed of six conserved DNA binding domains (DBDs). Deletion and mutational studies have identified a high-affinity DNA binding core in the central region of the 70 kDa subunit, composed of DBDs A and B. To define the roles of each DBD in DNA binding, monomeric and tandem DBD A and B domain chimeras were created and characterized. Individually, DBDs A and B have a very low intrinsic affinity for ssDNA. In contrast, tandem DBDs (AA, AB, BA, and BB) bind ssDNA with moderate to high affinity. The AA chimera had a much higher affinity for ssDNA than did the other tandem DBDs, demonstrating that DBD A has a higher intrinsic affinity for ssDNA than DBD B. The RPA-DNA interface is similar in both DBD A and DBD B. Mutational analysis was carried out to probe the relative contributions of the two domains to DNA binding. Mutation of polar residues in either core DBD resulted in a significant decrease in the affinity of the RPA complex for ssDNA. RPA complexes with pairs of mutated polar residues had lower affinities than those with single mutations. The decrease in affinity observed when polar mutations were combined suggests that multiple polar interactions contribute to the affinity of the RPA core for DNA. These results indicate that RPA-ssDNA interactions are the result of binding of multiple nonequivalent domains. Our data are consistent with a sequential binding model for RPA, in which DBD A is responsible for positioning and initial binding of the RPA complex while DBD A together with DBD B direct stable, high-affinity binding to ssDNA.