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.     

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.     

The origin recognition complex in silencing, cell cycle progression, and DNA replication.

This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5. The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest. At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication. The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins. Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication. The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes.     

ADP-binding to origin recognition complex of Saccharomyces cerevisiae

The origin recognition complex (ORC), a possible initiator of chromosomal DNA replication in eukaryotes, binds to ATP through its subunits Orc1p and Orc5p. Orc1p possesses ATPase activity. As for DnaA, the Escherichia coli initiator, the ATP-DnaA complex is active but the ADP-DnaA complex is inactive for DNA replication and, therefore, the ATPase activity of DnaA inactivates the ATP-DnaA complex to suppress the re-initiation of chromosomal DNA replication. We investigated ADP-binding to ORC by a filter-binding assay. The K(d) values for ADP-binding to wild-type ORC and to ORC-1A (ORC containing Orc1p with a defective Walker A motif) were less than 10nM, showing that Orc5p can bind to ADP with a high affinity, similar to ATP. ORC-5A (ORC containing Orc5p with a defective Walker A motif) did not bind to ADP, suggesting that the ADP-Orc1p complex is too unstable to be detected by the filter-binding assay. ADP dissociated more rapidly than ATP from wild-type ORC and ORC-1A. Origin DNA fragments did not stimulate ADP-binding to any type of ORC. In the presence of ADP, ORC could not bind to origin DNA in a sequence-specific manner. Thus, in eukaryotes, the ADP-ORC complex may be unable to initiate chromosomal DNA replication, and in this it resembles the ADP-DnaA complex in prokaryotes. However, overall control may be different. In eukaryotes, the ADP-ORC complex is unstable, suggesting that the ADP-ORC complex might rapidly become an ATP-ORC complex; whereas in prokaryotes, ADP remains bound to DnaA, keeping DnaA inactive, and preventing re-initiation for some periods.     

Analysis of origin recognition complex in saccharomyces cerevisiae by use of Degron mutants

Origin recognition complex (ORC), a six-protein complex (Orc1p-Orc6p), may deeply involve in initiation of chromosomal DNA replication. However, since most temperature-sensitive orc mutants of Saccharomyces cerevisiae show the accumulation of cells with nearly 2C DNA content, the exact stage at which ORC acts is not fully understood. In this study, we constructed a heat-inducible degron mutant for each ORC subunit. As well as each targeted subunit, other subunits of ORC were also rapidly degraded under non-permissive conditions. In the orc5 degron mutant, incubation under the non-permissive conditions caused accumulation of cells with nearly 2C DNA content, and phosphorylation of Rad53p. When Orc5p (ORC) is depleted, this inhibits G1/S transition and formation of a pre-replicative complex (pre-RC). For pre-RC to form, and G1/S transition to proceed, Orc5p (ORC) must be present in late G1, rather than early G1, or G2/M. Block and release experiments revealed that Orc5p (ORC) is not necessary for S and G2/M phase progression. We therefore propose that ORC is necessary for the G1/S transition and pre-RC formation, and accumulation of cells with nearly 2C DNA content seen in various orc mutants is due to inefficient pre-RC formation, and/or induction of checkpoint systems.     

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.     

ATP-dependent assembly of the human origin recognition complex

The origin recognition complex (ORC) was initially discovered in budding yeast extracts as a protein complex that binds with high affinity to autonomously replicating sequences in an ATP-dependent manner. We have cloned and expressed the human homologs of the ORC subunits as recombinant proteins. In contrast to other eukaryotic initiators examined thus far, assembly of human ORC in vitro is dependent on ATP binding. Mutations in the ATP-binding sites of Orc4 or Orc5 impair complex assembly, whereas Orc1 ATP binding is not required. Immunofluorescence staining of human cells with anti-Orc3 antibodies demonstrate cell cycle-dependent association with a nuclear structure. Immunoprecipitation experiments show that ORC disassembles as cells progress through S phase. The Orc6 protein binds directly to the Orc3 subunit and interacts as part of ORC in vivo. These data suggest that the assembly and disassembly of ORC in human cells is uniquely regulated and may contribute to restricting DNA replication to once in every cell division cycle.     

Dephosphorylation of Orc2 by protein phosphatase 1 promotes the binding of the origin recognition complex to chromatin

Phosphorylation of Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2–5) from human chromatin and replication origins. Dephosphorylation of the phosphorylated Orc2 by protein phosphatase 1 (PP1) is accompanied by the binding of the dissociated subunits to chromatin. Here we show that PP1 physically interacts with Orc2. The binding of PP1 to Orc2 and the dephosphorylation of Orc2 by PP1 occurred in a cell cycle-dependent manner through an interaction with 119-KSVSF-123, which is the consensus motif for the binding of PP1, of Orc2. The dephosphorylation of Orc2 by PP1 is required for the binding of Orc2 to chromatin. These results support that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin and replication origins for the subsequent round of the cell cycle.     

Yeast two-hybrid analysis of the origin recognition complex of Saccharomyces cerevisiae: interaction between subunits and identification of binding proteins

Origin recognition complex (ORC), a six-protein complex (Orc1p-6p), is the most likely initiator of chromosomal DNA replication in eukaryotes. Although ORC of Saccharomyces cerevisiae has been studied extensively from biochemical and genetic perspectives, its quaternary structure remains unknown. Previous studies suggested that ORC has functions other than DNA replication, such as gene silencing, but the molecular mechanisms of these functions have not been determined. In this study, we used yeast two-hybrid analysis to examine the interaction between ORC subunits and to search for ORC-binding proteins. As well as the known Orc4p-Orc5p interaction, we revealed strong interactions between Orc2p and Ord3p (2p-3p), Orc2p and Ord5p (2p-5p), Orc2p and Ord6p (2p-6p) and Orc3p and Ord6p (3p-6p) and weaker interactions between Orc1p and Ord4p (1p-4p), Orc3p and Ord4p (3p-4p), Orc2p and Ord3p (3p-5p) and Orc5p and Ord3p (5p-6p). These results suggest that 2p-3p-6p may form a core complex. Orc2p and Orc6p are phosphorylated in vivo, regulating initiation of DNA replication. However, replacing the phosphorylated amino acid residues with others that cannot be phosphorylated, or that mimic phosphorylation, did not affect subunit interactions. We also identified several proteins that interact with ORC subunits; Sir4p and Mad1p interact with Orc2p; Cac1p and Ykr077wp with Orc3p; Rrm3p and Swi6p with Orc5p; and Mih1p with Orc6p. We discuss roles of these interactions in functions of ORC.     

ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication

The origin recognition complex (ORC) is a six-subunit, ATP-regulated, DNA binding protein that is required for the formation of the prereplicative complex (pre-RC), an essential replication intermediate formed at each origin of DNA replication. In this study, we investigate the mechanism of ORC function during pre-RC formation and how ATP influences this event. We demonstrate that ATP hydrolysis by ORC requires the coordinate function of the Orc1 and Orc4 subunits. Mutations that eliminate ORC ATP hydrolysis do not support cell viability and show defects in pre-RC formation. Pre-RC formation involves reiterative loading of the putative replicative helicase, Mcm2-7, at the origin. Importantly, preventing ORC ATP hydrolysis inhibits this repeated Mcm2-7 loading. Our findings indicate that ORC is part of a helicase-loading molecular machine that repeatedly assembles Mcm2-7 complexes onto origin DNA and suggest that the assembly of multiple Mcm2-7 complexes plays a critical role in origin function.     

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.     

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.     

Subsets of human origin recognition complex (ORC) subunits are expressed in non-proliferating cells and associate with non-ORC proteins

The origin recognition complex (ORC) in yeast is a complex of six tightly associated subunits essential for the initiation of DNA replication. Human ORC subunits are nuclear in proliferating cells and in proliferative tissues like the testis, consistent with a role of human ORC in DNA replication. Orc2, Orc3, and Orc5 also are detected in non-proliferating cells like cardiac myocytes, adrenal cortical cells, and neurons, suggesting an additional role of these proteins in non-proliferating cells. Although Orc2-5 co-immunoprecipitate with each other under mild extraction conditions, a holo complex of the subunits is difficult to detect. When extracted under more stringent extraction conditions, several of the subunits co-immunoprecipitate with stoichiometric amounts of other unidentified proteins but not with any of the known ORC subunits. The variation in abundance of individual ORC subunits (relative to each other) in several tissues, expression of some subunits in non-proliferating tissues, and the absence of a stoichiometric complex of all the subunits in cell extracts indicate that subunits of human ORC in somatic cells might have activities independent of their role as a six subunit complex involved in replication initiation. Finally, all ORC subunits remain consistently nuclear, and Orc2 is consistently phosphorylated through all stages of the cell cycle, whereas Orc1 is selectively phosphorylated in mitosis.     

Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1

Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA.     

Characterization of an essential Orc2p-associated factor that plays a role in DNA replication.

The Saccharomyces cerevisiae Orc2 protein is a subunit of the origin recognition complex, ORC, which binds in a sequence-specific manner to yeast origins of DNA replication. With screens for orc2-1 synthetic lethal mutations and Orc2p two-hybrid interactors, a novel Orc2p-associated factor (Oaf1p) was identified. OAF1 is essential, its gene product is localized to the nucleus, and an oaf1 temperature-sensitive mutant arrests as large budded cells with a single nucleus. The mutant oaf1-2, isolated in the synthetic lethal screen, loses plasmids containing a single origin of DNA replication at a high rate, but it maintains plasmids carrying multiple potential origins of DNA replication. In addition, the OAF1 gene product tagged with the hemagglutinin antigen epitope binds to a DNA affinity column containing covalently linked tandem repeats of an essential origin element. These results suggest a role for OAFI in the initiation of DNA replication. Mutant alleles of cdc7 and cdc14 were also isolated in the orc2-1 synthetic lethal screen. Cdc7p, like Oaf1p, also interacts with Orc2p in two-hybrid assays.     

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.     

The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo

Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a bromo-adjacent homology (BAH) domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It did, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it was required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP-dependent plasmid DNA replication. Moreover, the BAH domain affected Orc1's ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin.     

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.