Analysis on Origin Recognition Complex containing Orc5p with defective Walker A Motif

Takahashi, Naoko; Yamaguchi, Yoshihiro; Yamairi, Fumiko; Makise, Masaki; Takenaka, Hitomi; Tsuchiya, Tomofusa; Mizushima, Tohru

doi:10.1074/jbc.m305531200

Cited by 16 publications

(27 citation statements)

References 42 publications

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“…Because Orc5-Ap showed decreased affinity for Orc4p by yeast two-hybrid analysis (36), one possibility is that the mutation in ORC-5A affects its interaction with Orc4p in a manner that is independent of ATP binding to Orc5p, which is responsible for its instability in cells. Supporting this notion, we have previously shown that overexpression of Orc4p suppresses the growth defect phenotype of the orc5-A strain (33) but not that of the orc2-5d strain (data not shown in this paper). One remaining unsolved question is whether the defect in ATP binding to Orc5p is responsible for the phenotype exhibited by the orc2-5d strain, such as a slow G 1 -S transition, induction of phosphorylation of Rad53p, and inefficient loading of MCM onto chromatin.…”

Section: Discussionsupporting

confidence: 58%

“…Because our previous yeast two-hybrid analysis showed that Orc2p has a strong interaction with Orc5p (36), it is possible that phosphorylation of Orc2p affects the structure of Orc5p resulting in it losing its affinity for ATP. We recently reported that ORC-5A (ORC containing a mutant Orc5p with a defective Walker A motif) is unstable in cells due to degradation by the ubiquitin-proteasome system (33,34). However, we have shown here that ORC2-5D is stable in cells.…”

Section: Discussionmentioning

confidence: 42%

“…The binding of ATP to Orc1p, but not to Orc5p, is essential for the specific binding of ORC to origin DNA (28,29). ATP binding to Orc5p increases the affinity of Orc1p for ATP in vitro (29) and is important for maintaining the stability of ORC in vivo (33,34). However, a link between phosphorylation of ORC and its ATP binding activity has not been previously established.…”

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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: Discussionsupporting

confidence: 58%

Section: Discussionmentioning

confidence: 42%

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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“…Orc5 is one of the ORC subunits, and ATP binding to Orc5 is involved in efficient ORC formation (Siddiqui and Stillman, 2007;Takahashi et al, 2004). Orc5 has also been implicated in silencing at the HML and MHR loci in yeast and in heterochromatin organization in human cells (Dillin and Rine, 1997;Prasanth et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

Orc5 induces large-scale chromatin decondensation in a GCN5-dependent manner

Giri

Chakraborty

Sathyan

et al. 2015

Journal of Cell Science

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In eukaryotes, origin recognition complex (ORC) proteins establish the pre-replicative complex ( preRC) at the origins, and this is essential for the initiation of DNA replication. Open chromatin structures regulate the efficiency of preRC formation and replication initiation. However, the molecular mechanisms that control chromatin structure, and how the preRC components establish themselves on the chromatin remain to be understood. In human cells, the ORC is a highly dynamic complex with many separate functions attributed to sub-complexes or individual subunits of the ORC, including heterochromatin organization, telomere and centromere function, centrosome duplication and cytokinesis. We demonstrate that human Orc5, unlike other ORC subunits, when ectopically tethered to a chromatin locus, induces large-scale chromatin decondensation, predominantly during G1 phase of the cell cycle. Orc5 associates with the H3 histone acetyl transferase GCN5 (also known as KAT2A), and this association enhances the chromatin-opening function of Orc5. In the absence of Orc5, histone H3 acetylation is decreased at the origins. We propose that the ability of Orc5 to induce chromatin unfolding during G1 allows the establishment of the preRC at the origins.

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“…In S. cerevisiae and Drosophila the ATP-binding activity of Orc1 is essential and regulates DNA binding. Although not essential, mutations in the S. cerevisiae Orc5 ATP-binding motif cause defects in the apparent complex stability (Takahashi et al, 2004). In contrast, mutations of the Orc1, Orc4 or Orc5 ATP-binding motifs inhibit the ability of human ORC to activate replication in ORC-depleted Xenopus egg extracts (Giordano-Coltart et al, 2005).…”

Section: Origin Recognition Complexmentioning

confidence: 99%