Kinetics of ATP binding to the origin recognition complex of Saccharomyces cerevisiae

Origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, binds specifically to ATP through two of its subunits (Orc1p and Orc5p). In this study, we investigated the kinetics of ATP binding to ORC by a filter binding assay. The Kd values for the ATP of wild-type ORC and ORC-1A (mutant ORC containing Orc1p with a defective Walker A motif) were less than 10 nm, suggesting that the affinity of Orc5p for ATP is very high. On the other hand, the Kd values for the ATP of ORC-5A (mutant ORC containing Orc5p with a defective Walker A motif) was much higher (about 1.5 microm), suggesting that the affinity of Orc1p for ATP is relatively low in the absence of origin DNA. ATP dissociated more rapidly from its complex with ORC-5A than from its complex with ORC-1A, suggesting that the ATP-Orc5p complex is more stable than ATP-Orc1p complex. Origin DNA fragments decreased the Kd value of ORC-5A for ATP and stabilized the complex of ATP with ORC-5A. Wild-type ORC, ORC-1A, and ORC-5A required different concentrations of ATP for specific binding to origin DNA. All of these results imply that ATP binding to Orc5p, ATP binding to Orc1p, and origin DNA binding to ORC are co-operatively regulated, which may be important for the initiation of DNA replication.     

Rapid Exchange of Bound ADP on the Staphylococcus aureus Replication Initiation Protein DnaA

In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in Gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but not ADP by a mechanism independent of ATP hydrolysis. Unlike E. coli DnaA, binding of ADP to S. aureus DnaA was unstable. As a result, at physiological concentrations of ATP, ADP bound to S. aureus DnaA was rapidly exchanged for ATP, thereby regenerating the ability of DnaA to form the open complex in vitro. Therefore, we examined whether formation of ADP-DnaA participates in suppression of replication initiation in vivo. Induction of the R318H mutant of the AAA+ sensor 2 protein, which has decreased intrinsic ATPase activity, caused over-initiation of chromosome replication in S. aureus, suggesting that formation of ADP-DnaA suppresses the initiation step in S. aureus. Together with the biochemical features of S. aureus DnaA, the weak ability to convert ATP-DnaA into ADP-DnaA and the instability of ADP-DnaA, these results suggest that there may be unidentified system(s) for reducing the cellular ratio of ATP-DnaA to ADP-DnaA in S. aureus and thereby delaying the re-initiation of DNA replication.     

Stable nucleotide binding to DnaA requires a specific glutamic acid residue within the AAA+ box II motif

In complex with ATP, but not ADP, DnaA protein multimers unwind a specific region of duplex DNA within the chromosomal replication origin, oriC, triggering a series of reactions that result in initiation of DNA replication. Following replication initiation, ATP hydrolysis, which is coupled to DNA replication, results in the generation of initiation-incompetent ADP-DnaA. Suppression of overinitiation of replication requires that ADP-DnaA complexes be stably maintained until the next round of replication. Thus, the functional and structural requirements that ensure stable nucleotide binding to DnaA are crucial for proper regulation of replication. Here, we demonstrate that Glu143 of DnaA, located within the AAA+ box II N-linker motif, is a key residue involved in stable nucleotide binding. A Glu143 substitution variant of DnaA (DnaA E143A) bound to ADP on ice with an affinity similar to wild-type DnaA, but the resultant ADP-DnaA E143A complex was more labile at 37 °C than wild-type ADP-DnaA complexes. Consistent with this, conversion of ADP-DnaA E143A to ATP-DnaA E143A was stimulated at 37°C in the presence of ATP, which also stimulated replication of a minichromosome in an in vitro reconstitution reaction. Expression of DnaA E143A in vivo inhibited cell growth in an oriC-dependent manner, suggesting that DnaA E143A caused over-initiation of replication, consistent with the in vitro results. Glu is a highly conserved residue at the corresponding position of γ-proteobacterial DnaA orthologs. Our finding of the novel role for the DnaA N-linker region may represent a conserved function of this motif among those DnaA orthologs.     

Analysis on origin recognition complex containing Orc5p with defective Walker A motif

Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37 degrees C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process.     

Analysis of mutant origin recognition complex with reduced ATPase activity in vivo and in vitro

In eukaryotes, ORC (origin recognition complex), a six-protein complex, is the most likely initiator of chromosomal DNA replication. ORC belongs to the AAA(+) (ATPases associated with a variety of cellular activities) family of proteins and has intrinsic ATPase activity derived from Orc1p, one of its subunits. To reveal the role of this ATPase activity in Saccharomyces cerevisiae (baker's yeast) ORC, we mutated the Orc1p sensor 1 and sensor 2 regions, which are important for ATPase activity in AAA(+) proteins. Plasmid-shuffling analysis revealed that Asn(600), Arg(694) and Arg(704) are essential for the function of Orc1p. In yeast cells, overexpression of Orc1R694Ep inhibited growth, caused inefficient loading of MCM (mini-chromosome maintenance complex of proteins) and slowed the progression of S phase. In vitro, purified ORC-1R [ORC with Orc1R694Ep (Orc1p Arg(694)-->Glu mutant)] has decreased ATPase activity in the presence or absence of origin DNA. However, other activities (ATP binding and origin DNA binding) were indistinguishable from those of wild-type ORC. The present study showed that Arg(694) of the Orc1p subunit is important for the ATPase activity of ORC and suggests that this ATPase activity is required for efficient MCM loading on to origin DNA and for progression of S phase.     

Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli

The ATP-bound but not the ADP-bound form of DnaA protein is active for replication initiation at the Escherichia coli chromosomal origin. The hydrolysis of ATP bound to DnaA is accelerated by the sliding clamp of DNA polymerase III loaded on DNA. Using a culture of randomly dividing cells, we now have evidence that the cellular level of ATP-DnaA is repressed to only approximately 20% of the total DnaA molecules, in a manner depending on DNA replication. In a synchronized culture, the ATP-DnaA level showed oscillation that has a temporal increase around the time of initiation, and decreases rapidly after initiation. Production of ATP-DnaA depended on concomitant protein synthesis, but not on SOS response, Dam or SeqA. Regeneration of ATP-DnaA from ADP-DnaA was also observed. These results indicate that the nucleotide form shifts of DnaA are tightly linked with an epistatic cell cycle event and with the chromosomal replication system.     

Interaction between ORC and Cdt1p of Saccharomyces cerevisiae

Origin recognition complex (ORC), a six-protein complex, is the most likely initiator of chromosomal DNA replication in eukaryotes. Throughout the cell cycle, ORC binds to chromatin at origins of DNA replication and functions as a 'landing pad' for the binding of other proteins, including Cdt1p, to form a prereplicative complex. In this study, we used yeast two-hybrid analysis to examine the interaction between Cdt1p and every ORC subunit. We observed potent interaction with Orc6p, and weaker interaction with Orc2p and Orc5p. Coimmunoprecipitation assay confirmed that Cdt1p interacted with Orc6p, as well as with Orc1p and Orc2p. We mapped the C-terminal region, and a middle region of Orc6p (amino acids residues 394-435, and 121-175, respectively), as important for interaction with Cdt1p. Cdt1p was purified to examine its direct interaction with ORC, and its effect on the activity of ORC. Glutathione-S-transferase pull-down analysis revealed that Cdt1p binds directly to ORC. Cdt1p neither bound to origin DNA and ATP nor affected ORC-binding to origin DNA and ATP. These results suggest that interaction of Cdt1p with ORC is involved in the formation of the prereplicative complex, rather than in regulation of the activity of ORC.     

Mechanism for the degradation of origin recognition complex containing Orc5p with a defective Walker A motif and its suppression by over-production of Orc4p in yeast cells

Orc5p is one of six subunits constituting the ORC (origin recognition complex), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell-cycle arrest at G2/M and degradation of ORC at high temperatures (37 degrees C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes [Takahashi, Yamaguchi, Yamairi, Makise, Takenaka, Tsuchiya and Mizushima (2004) J. Biol. Chem. 279, 8469-8477]. In the present study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tan1-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tan1-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP binding to Orc5p is required for efficient interaction with Orc4p and that, in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why over-production of Orc4p suppresses the orc5-A strain's phenotype.     

The chromosome origin of Escherichia coli stabilizes DnaA protein during rejuvenation by phospholipids.

DnaA protein (the initiator protein) binds and clusters at the four DnaA boxes of the Escherichia coli chromosomal origin (oriC) to promote the strand opening for DNA replication. DnaA protein activity depends on the tight binding of ATP; the ADP form of DnaA protein, generated by hydrolysis of the bound ATP, is inactive. Rejuvenation of ADP-DnaA protein, by replacement with ATP, is catalyzed by acidic phospholipids in a highly fluid bilayer. We find that interaction of DnaA protein with oriC DNA is needed to stabilize DnaA protein during this rejuvenation process. Whereas DnaA protein bound to oriC DNA responds to phospholipids, free DnaA protein is inactivated by phospholipids and then fails to bind oriC. Furthermore, oriC DNA facilitates the high affinity binding of ATP to DnaA protein during treatment with phospholipids. A significant portion of the DnaA protein associated with oriC DNA can be replaced by the ADP form of the protein, suggesting that all of the DnaA protein bound to oriC DNA need not be rejuvenated between rounds of replication.     

The origin recognition complex protein family

Origin recognition complex (ORC) proteins were first discovered as a six-subunit assemblage in budding yeast that promotes the initiation of DNA replication. Orc1-5 appear to be present in all eukaryotes, and include both AAA+ and winged-helix motifs. A sixth protein, Orc6, shows no structural similarity to the other ORC proteins, and is poorly conserved between budding yeast and most other eukaryotic species. The replication factor Cdc6 has extensive sequence similarity with Orc1 and phylogenetic analysis suggests the genes that encode them may be paralogs. ORC proteins have also been found in the archaea, and the bacterial DnaA replication protein has ORC-like functional domains. In budding yeast, Orc1-6 are bound to origins of DNA replication throughout the cell cycle. Following association with Cdc6 in G1 phase, the sequential hydrolysis of Cdc6 - then ORC-bound ATP loads the Mcm2-7 helicase complex onto DNA. Localization of ORC subunits to the kinetochore and centrosome during mitosis and to the cleavage furrow during cytokinesis has been observed in metazoan cells and, along with phenotypes observed following knockdown with short interfering RNAs, point to additional roles at these cell-cycle stages. In addition, ORC proteins function in epigenetic gene silencing through interactions with heterochromatin factors such as Sir1 in budding yeast and HP1 in higher eukaryotes. Current avenues of research have identified roles for ORC proteins in the development of neuronal and muscle tissue, and are probing their relationship to genome integrity.     

Genetic analysis of human Orc2 reveals specific domains that are required in vivo for assembly and nuclear localization of the origin recognition complex

Eukaryotic DNA replication begins with the binding of a six subunit origin recognition complex (ORC) to DNA. To study the assembly and function of mammalian ORC proteins in their native environment, HeLa cells were constructed that constitutively expressed an epitope-tagged, recombinant human Orc2 subunit that had been genetically altered. Analysis of these cell lines revealed that Orc2 contains a single ORC assembly domain that is required in vivo for interaction with all other ORC subunits, as well as two nuclear localization signals (NLSs) that are required for ORC accumulation in the nucleus. The recombinant Orc2 existed in the nucleus either as an ORC-(2-5) or ORC-(1-5) complex; no other combinations of ORC subunits were detected. Moreover, only ORC-(1-5) was bound to the chromatin fraction, suggesting that Orc1 is required in vivo to load ORC-(2-5) onto chromatin. Surprisingly, recombinant Orc2 suppressed expression of endogenous Orc2, revealing that mammalian cells limit the intracellular level of Orc2, and thereby limit the amount of ORC-(2-5) in the nucleus. Because this suppression required only the ORC assembly and NLS domains, these domains appear to constitute the functional domain of Orc2.     

Formation of an ATP-DnaA-specific initiation complex requires DnaA Arginine 285, a conserved motif in the AAA+ protein family

Escherichia coli DnaA protein, a member of the AAA+ superfamily, initiates replication from the chromosomal origin oriC in an ATP-dependent manner. Nucleoprotein complex formed on oriC with the ATP-DnaA multimer but not the ADP-DnaA multimer is competent to unwind the oriC duplex. The oriC region contains ATP-DnaA-specific binding sites termed I2 and I3, which stimulate ATP-DnaA-dependent oriC unwinding. In this study, we show that the DnaA R285A mutant is inactive for oriC replication in vivo and in vitro and that the mutation is associated with specific defects in oriC unwinding. In contrast, activities of DnaA R285A are sustained in binding to the typical DnaA boxes and to ATP and ADP, formation of multimeric complexes on oriC, and loading of the DnaB helicase onto single-stranded DNA. Footprint analysis of the DnaA-oriC complex reveals that the ATP form of DnaA R285A does not interact with ATP-DnaA-specific binding sites such as the I sites. A subgroup of DnaA molecules in the oriC complex must contain the Arg-285 residue for initiation. Sequence and structural analyses suggest that the DnaA Arg-285 residue is an arginine finger, an AAA+ family-specific motif that recognizes ATP bound to an adjacent subunit in a multimeric complex. In the context of these and previous results, the DnaA Arg-285 residue is proposed to play a unique role in the ATP-dependent conformational activation of an initial complex by recognizing ATP bound to DnaA and by modulating the structure of the DnaA multimer to allow interaction with ATP-DnaA-specific binding sites in the complex.     

Site-Specific DNA Binding of the Schizosaccharomyces pombe Origin Recognition Complex Is Determined by the Orc4 Subunit

The mechanism by which origin recognition complexes (ORCs) identify replication origins was investigated using purified Orc proteins from Schizosaccharomyces pombe. Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, -2, -3, -5, and -6 proteins (ORC-5) altered neither Orc4p binding to origin DNA nor Orc4p protection of specific sequences. ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit.     

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.     

A novel role for cAMP in the control of the activity of the E. coli chromosome replication initiator protein, DnaA

DnaA protein interacts with cAMP with a KD of 1 microM. This interaction stimulates DnaA protein binding to the chromosome replication origin (oriC) and the mioC promoter region, protects DnaA protein from thermal inactivation, releases ADP but not ATP bound to DnaA protein, and restores normal DNA replication activity and ATPase activity in inactive ADP-DnaA protein preparations. A model is proposed in which cellular cAMP levels govern the replication activity of DnaA protein by promoting the recycling of the inactive ADP-DnaA protein form into the active ATP form.     

Reactivation of DnaA by DNA sequence-specific nucleotide exchange in vitro

In Escherichia coli, ATP-bound DnaA protein can initiate chromosomal replication. After initiation, DnaA-ATP is hydrolyzed by interactions with a complex containing a replicase subunit to yield the inactive ADP-DnaA. However, the mechanisms which regenerate ATP-DnaA from ADP-DnaA are not well understood. We report here that a 70-bp DNA segment promotes exchange of the DnaA-bound nucleotide in a sequence-specific manner, thus reactivating the initiation function of DnaA in vitro. This segment contains a typical DnaA-binding 9-mer motif, the DnaA box, and two DnaA box-like sequences. The presence and precise composition of these three motifs are required for the DnaA-reactivating activity, which suggests that a highly ordered complex which includes multimeric DnaA molecules is formed for isomerization of DnaA. We named this DNA segment DARS, for DnaA-reactivating sequence. The role of DARS in regulation of DnaA function in vivo is discussed.     

Membrane-catalyzed nucleotide exchange on DnaA. Effect of surface molecular crowding

DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3'-O-(N-methylantraniloyl)-5'-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.