Linkage between phosphorylation of the origin recognition complex and its ATP binding activity in Saccharomyces cerevisiae

Masaki Makise, Masaya Takehara, Akihiko Kuniyasu, Nanako Matsui, Hitoshi Nakayama, Tohru Mizushima

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The initiation of chromosomalDNAreplication 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 phosphomimetic mutants of Orc2p or Orc6p and found that expression of a Ser-188 mutant of Orc2p (Orc2-5Dp) delays G1-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 G1 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.

Original languageEnglish
Pages (from-to)3396-3407
Number of pages12
JournalJournal of Biological Chemistry
Volume284
Issue number6
DOIs
Publication statusPublished - 2009 Feb 6
Externally publishedYes

Fingerprint

Origin Recognition Complex
Phosphorylation
Cyclin-Dependent Kinases
Yeast
Saccharomyces cerevisiae
Cell Cycle
Adenosine Triphosphate
Cells
Minichromosome Maintenance Proteins
Adenine Nucleotides
DNA
G1 Phase
Cell Cycle Checkpoints
DNA Replication
S Phase
Cell Division
Yeasts
Genome
Antibodies
Demonstrations

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Linkage between phosphorylation of the origin recognition complex and its ATP binding activity in Saccharomyces cerevisiae. / Makise, Masaki; Takehara, Masaya; Kuniyasu, Akihiko; Matsui, Nanako; Nakayama, Hitoshi; Mizushima, Tohru.

In: Journal of Biological Chemistry, Vol. 284, No. 6, 06.02.2009, p. 3396-3407.

Research output: Contribution to journalArticle

Makise, Masaki ; Takehara, Masaya ; Kuniyasu, Akihiko ; Matsui, Nanako ; Nakayama, Hitoshi ; Mizushima, Tohru. / Linkage between phosphorylation of the origin recognition complex and its ATP binding activity in Saccharomyces cerevisiae. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 6. pp. 3396-3407.
@article{0853a547c29a4b8e99dc09bf3e746287,
title = "Linkage between phosphorylation of the origin recognition complex and its ATP binding activity in Saccharomyces cerevisiae",
abstract = "The initiation of chromosomalDNAreplication 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 phosphomimetic mutants of Orc2p or Orc6p and found that expression of a Ser-188 mutant of Orc2p (Orc2-5Dp) delays G1-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 G1 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.",
author = "Masaki Makise and Masaya Takehara and Akihiko Kuniyasu and Nanako Matsui and Hitoshi Nakayama and Tohru Mizushima",
year = "2009",
month = "2",
day = "6",
doi = "10.1074/jbc.M804293200",
language = "English",
volume = "284",
pages = "3396--3407",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "6",

}

TY - JOUR

T1 - Linkage between phosphorylation of the origin recognition complex and its ATP binding activity in Saccharomyces cerevisiae

AU - Makise, Masaki

AU - Takehara, Masaya

AU - Kuniyasu, Akihiko

AU - Matsui, Nanako

AU - Nakayama, Hitoshi

AU - Mizushima, Tohru

PY - 2009/2/6

Y1 - 2009/2/6

N2 - The initiation of chromosomalDNAreplication 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 phosphomimetic mutants of Orc2p or Orc6p and found that expression of a Ser-188 mutant of Orc2p (Orc2-5Dp) delays G1-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 G1 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.

AB - The initiation of chromosomalDNAreplication 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 phosphomimetic mutants of Orc2p or Orc6p and found that expression of a Ser-188 mutant of Orc2p (Orc2-5Dp) delays G1-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 G1 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.

UR - http://www.scopus.com/inward/record.url?scp=63649083216&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=63649083216&partnerID=8YFLogxK

U2 - 10.1074/jbc.M804293200

DO - 10.1074/jbc.M804293200

M3 - Article

C2 - 19068484

AN - SCOPUS:63649083216

VL - 284

SP - 3396

EP - 3407

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 6

ER -