Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: Purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus

Hirokazu Nishida, Shigeki Matsumiya, Daisuke Tsuchiya, Yoshizumi Ishino, Kosuke Morikawa

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Replicative DNA polymerase interacts with processivity factors, the β-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 Å resolution using synchrotron radiation. The crystals belong to space group P21212, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 Å.

Original languageEnglish
Pages (from-to)253-256
Number of pages4
JournalActa Crystallographica Section F: Structural Biology and Crystallization Communications
Volume62
Issue number3
DOIs
Publication statusPublished - Jun 21 2006

Fingerprint

Pyrococcus furiosus
Proliferating Cell Nuclear Antigen
antigens
DNA-Directed DNA Polymerase
Crystallization
purification
Purification
deoxyribonucleic acid
monomers
Monomers
crystallization
proteins
Proteins
Archaeal DNA
DNA Polymerase III
rings
Amino Acid Motifs
Crystals
Synchrotrons
crystals

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Genetics
  • Condensed Matter Physics

Cite this

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title = "Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: Purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus",
abstract = "Replicative DNA polymerase interacts with processivity factors, the β-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 {\AA} resolution using synchrotron radiation. The crystals belong to space group P21212, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 {\AA}.",
author = "Hirokazu Nishida and Shigeki Matsumiya and Daisuke Tsuchiya and Yoshizumi Ishino and Kosuke Morikawa",
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T2 - Purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus

AU - Nishida, Hirokazu

AU - Matsumiya, Shigeki

AU - Tsuchiya, Daisuke

AU - Ishino, Yoshizumi

AU - Morikawa, Kosuke

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AB - Replicative DNA polymerase interacts with processivity factors, the β-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 Å resolution using synchrotron radiation. The crystals belong to space group P21212, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 Å.

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