ADP-binding to Origin Recognition Complex of Saccharomyces cerevisiae

Takenaka, Hitomi; Makise, Masaki; Kuwae, Wakako; Takahashi, Naoko; Tsuchiya, Tomofusa; Mizushima, Tohru

doi:10.1016/j.jmb.2004.04.045

Cited by 12 publications

(13 citation statements)

References 38 publications

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“…6C, in the presence of ATP, wild-type ORC and ORC-5A but not ORC-1A bound to wild-type ARS1 but not to mutant ars1/A Ϫ B1 DNA fragments. All of these ORCs did not bind to any DNA fragments in the presence of ADP, as described previously (32). The DNA binding properties of ORC2-5D were indistinguishable from that of wild-type ORC even in the gel electrophoretic mobility shift assay (Fig.…”

Section: Ymm10-3 (Orc2)supporting

confidence: 67%

“…Gel Electrophoretic Mobility Shift Assay-A gel electrophoretic mobility shift assay was performed as described (32,43) with some modifications. ORCs were incubated with adenine nucleotides for 5 min at 30°C and with radiolabeled wildtype ARS1 or mutant ars1/A Ϫ…”

Section: B1mentioning

confidence: 99%

“…Orc1p, but not Orc5p, has ATPase activity, which stimulates the loading of MCM onto origins (30,31). Orc5p, but not Orc1p, can bind ADP (32). The binding of ATP to Orc1p, but not to Orc5p, is essential for the specific binding of ORC to origin DNA (28,29).…”

mentioning

confidence: 99%

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Linkage between Phosphorylation of the Origin Recognition Complex and Its ATP Binding Activity in Saccharomyces cerevisiae

Makise

Takehara

Kuniyasu

et al. 2009

Journal of Biological Chemistry

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The initiation of chromosomal DNA replication is tightly regulated to achieve genome replication just once per cell cycle and cyclin-dependent kinase (CDK) plays an important role in this process. Adenine nucleotides that bind to the origin recognition complex (ORC) are also suggested to be involved in this process. Of the six subunits of the Saccharomyces cerevisiae ORC (Orc1-6p), both Orc1p and Orc5p have ATP binding activity, and both Orc2p and Orc6p are phosphorylated by CDK in cells. In this study we constructed a series of yeast strains expressing phospho-mimetic mutants of Orc2p or Orc6p and found that expression of a Ser-188 mutant of Orc2p (Orc2-5Dp) delays G 1 -S transition and S phase progression and causes the accumulation of cells with 2C DNA content. Using antibody that specifically recognizes Ser-188-phosphorylated Orc2p, we showed that Ser-188 is phosphorylated by CDK in a cell cycle-regulated manner. Expression of Orc2-5Dp caused phosphorylation of Rad53p and inefficient loading of the six minichromosome maintenance proteins. These results suggest that the accumulation of cells with 2C DNA content is due to inefficient origin firing and induction of the cell cycle checkpoint response and that dephosphorylation of Ser-188 of Orc2p in late M or G 1 phase may be involved in pre-RC formation. In vitro, a purified mutant ORC containing Orc2-5Dp lost Orc5p ATP binding activity. This is the first demonstration of a link between phosphorylation of the ORC and its ability to bind ATP, which may be important for the cell cycle-regulated initiation of DNA replication.The initiation of chromosomal DNA replication is tightly regulated to replicate the genome just once per cell cycle. To achieve this, both induction of initiation at the G 1 -S boundary and inhibition of initiation in other phases of the cell cycle are required. The mechanisms governing this regulation in eukaryotes have been studied the most extensively in budding yeast (Saccharomyces cerevisiae), and we describe mostly events in budding yeast in this paper otherwise noticed. Cyclindependent protein kinases (CDKs) 2 play essential roles in both the induction and inhibition of initiation; low CDK activity in late M and G 1 phases is required to prepare for initiation of DNA replication, and high CDK activity in S, G 2 , and early M phases is required for suppression of re-initiation of DNA replication before cell division. This high CDK activity is also involved in initiation of DNA replication at the G 1 -S boundary (1-4).Cell cycle-regulated formation of protein complexes on origins of chromosomal DNA replication is a key step in regulation of the initiation of DNA replication. In G 1 phase (under low CDK activity), a protein complex called the "pre-replication complex (pre-RC)" is formed on each origin. The pre-RC contains several proteins including the origin recognition complex (ORC), Cdc6p, Cdt1p, and the six minichromosome maintenance proteins (MCM), Mcm2-7p. The ORC was originally identified as a six-protein complex that specifically b...

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Section: Ymm10-3 (Orc2)supporting

confidence: 67%

Section: B1mentioning

confidence: 99%

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Linkage between Phosphorylation of the Origin Recognition Complex and Its ATP Binding Activity in Saccharomyces cerevisiae

Makise

Takehara

Kuniyasu

et al. 2009

Journal of Biological Chemistry

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“…In eukaryotes, initiation of replication by the yeast origin recognition complex (Orc) depends on ATP hydrolysis (63, 64). The ADP-bound form of Orc1p is thought to dissociate the initiation complex and promote the replication process by a mechanical process rather than acting as a suppressor replication initiation (65). Because the ATPase-deficient R318H mutant of S. aureus DnaA caused excessive replication initiation, like E. coli DnaA, it appears to act as a regulator of replication initiation rather than by mechanically influencing replication.…”

Section: Discussionmentioning

confidence: 99%

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

Kurokawa

Mizumura

Takaki

et al. 2009

Journal of Biological Chemistry

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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 ATPDnaA to ADP-DnaA in S. aureus and thereby delaying the reinitiation of DNA replication.The initiation step of chromosome replication is an important set point for coordinating cell growth and chromosomal replication, and it is tightly regulated so that it takes place only once per cell cycle. DNA replication in bacteria starts when the cell mass reaches an appropriate size and at the appropriate time, which is controlled in part by the quantity and activity of initiator DnaA protein (1-3). In Escherichia coli, DnaA binds to 9-mer DnaA boxes in oriC, the origin of chromosome replication, and melts the duplex at AT-rich 13-mers adjacent to oriC. DnaA then directs loading of the DnaB replicative helicase, which depends on the DnaC helicase loader and priming by the DnaG primase. This is followed by the synthesis of DNA by the DNA polymerase III holoenzyme (4).DnaA is composed of four domains: N-terminal domains I and II are involved in DnaA oligomerization and DnaADnaB interaction, and they show a diversity of sequence among bacteria. Domain III contains AAAϩ motifs, and the C-terminal domain IV mediates DNA binding (3, 5). Biochemical and genetic studies in E. coli have revealed adenine nucleotide binding-mediated control of the DnaA activity (6 -11). Specifically, ATP-bound DnaA actively promotes replication initiation, whereas the ADP-bound form is inert (6, 7). ATP promotes the self-assembly of DnaA at replication origins via a conformational change to form a righthanded helical filament (8, 9). In E. coli, 70% to 80% of DnaA exists as the inactive ADP-bound form, and active ATPDnaA levels increase during the initiation ...

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“…In addition to the structural similarities of origin recognition proteins found in both systems (Giraldo, 2003), the common theme of regulation by ATP binding and ATPase activity has extended to yeast and mammalian origin recognition complexes (ORCs) (Baker and Bell, 1998;Davey et al, 2002;Takenaka et al, 2004). Furthermore, recent studies suggest that single-stranded DNA plays a role in ORC protein binding and stimulation of ATPase regulatory activity .…”

Section: Beyond E Coli?mentioning

confidence: 99%

Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding

Leonard

Grimwade

2005

Molecular Microbiology

107

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SummaryTriggering new rounds of chromosomal DNA replication during the bacterial cell cycle is exquisitely regulated, ensuring both proper timing and one round per cycle stringency. A critical first step is stable unwinding of oriC , the chromosomal replication origin, by multiprotein orisome complexes comprising the AAA + initiator DnaA and modulator proteins that bend DNA. Recently identified oriC -DnaA interactions in Escherichia coli raise important questions regarding the molecular mechanisms that regulate origin unwinding in bacteria. We describe staged binding of E. coli origin recognition proteins and suggest an unwinding switch based on interactions between DnaA-ATP and specialized oriC sites that must be filled during orisome assembly. By focusing multiple regulatory pathways on only a few key oriC DNA-protein interactions, this model includes an efficient way to control unwinding followed by orisome inactivation during the cell cycle. Future studies will determine whether this regulatory scheme is correct and whether it is generally applicable to other bacterial types.

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ADP-binding to Origin Recognition Complex of Saccharomyces cerevisiae

Cited by 12 publications

References 38 publications

Linkage between Phosphorylation of the Origin Recognition Complex and Its ATP Binding Activity in Saccharomyces cerevisiae

Linkage between Phosphorylation of the Origin Recognition Complex and Its ATP Binding Activity in Saccharomyces cerevisiae

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

Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding

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