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

Kazuya Matsuda, Masaki Makise, Yoshihiro Sueyasu, Masaya Takehara, Teita Asano, Tohru Mizushima

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1263-1269
Number of pages7
JournalFEMS Yeast Research
Volume7
Issue number8
DOIs
Publication statusPublished - 2007 Dec
Externally publishedYes

Fingerprint

Origin Recognition Complex
Saccharomyces cerevisiae
Carrier Proteins
Yeasts
DNA Replication
Gene Silencing
Eukaryota
Molecular Biology
Proteins
Phosphorylation
Amino Acids

Keywords

  • DNA replication
  • Origin recognition complex
  • Subunits
  • Yeast two-hybrid analysis

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Microbiology
  • Infectious Diseases

Cite this

Yeast two-hybrid analysis of the origin recognition complex of Saccharomyces cerevisiae : Interaction between subunits and identification of binding proteins. / Matsuda, Kazuya; Makise, Masaki; Sueyasu, Yoshihiro; Takehara, Masaya; Asano, Teita; Mizushima, Tohru.

In: FEMS Yeast Research, Vol. 7, No. 8, 12.2007, p. 1263-1269.

Research output: Contribution to journalArticle

Matsuda, Kazuya ; Makise, Masaki ; Sueyasu, Yoshihiro ; Takehara, Masaya ; Asano, Teita ; Mizushima, Tohru. / Yeast two-hybrid analysis of the origin recognition complex of Saccharomyces cerevisiae : Interaction between subunits and identification of binding proteins. In: FEMS Yeast Research. 2007 ; Vol. 7, No. 8. pp. 1263-1269.
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T1 - Yeast two-hybrid analysis of the origin recognition complex of Saccharomyces cerevisiae

T2 - Interaction between subunits and identification of binding proteins

AU - Matsuda, Kazuya

AU - Makise, Masaki

AU - Sueyasu, Yoshihiro

AU - Takehara, Masaya

AU - Asano, Teita

AU - Mizushima, Tohru

PY - 2007/12

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AB - 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.

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KW - Subunits

KW - Yeast two-hybrid analysis

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