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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

A stable stoichiometric complex of archaeal DNA polymerase with proliferating cell nuclear antigen (PCNA) was formed using a PCNA monomer mutant and the complex was successfully crystallized. 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 P2{sub 1}2{sub 1}2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 Å.

Authors:
; ;  [1];  [2];  [1]
  1. Department of Structural Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka 565-0874 (Japan)
  2. Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka-shi, Fukuoka 812-8581 (Japan)
Publication Date:
OSTI Identifier:
22356277
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 3; Other Information: PMCID: PMC2197164; PMID: 16511315; PUBLISHER-ID: bw5125; OAI: oai:pubmedcentral.nih.gov:2197164; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALS; DNA; IN VITRO; MOLECULES; MONOMERS; RESOLUTION; RINGS; SPACE GROUPS; SYNCHROTRON RADIATION

Citation Formats

Nishida, Hirokazu, Matsumiya, Shigeki, Tsuchiya, Daisuke, Ishino, Yoshizumi, and Morikawa, Kosuke, E-mail: morikako@protein.osaka-u.ac.jp. 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. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309106004362.
Nishida, Hirokazu, Matsumiya, Shigeki, Tsuchiya, Daisuke, Ishino, Yoshizumi, & Morikawa, Kosuke, E-mail: morikako@protein.osaka-u.ac.jp. 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. United Kingdom. doi:10.1107/S1744309106004362.
Nishida, Hirokazu, Matsumiya, Shigeki, Tsuchiya, Daisuke, Ishino, Yoshizumi, and Morikawa, Kosuke, E-mail: morikako@protein.osaka-u.ac.jp. Wed . "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". United Kingdom. doi:10.1107/S1744309106004362.
@article{osti_22356277,
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},
author = {Nishida, Hirokazu and Matsumiya, Shigeki and Tsuchiya, Daisuke and Ishino, Yoshizumi and Morikawa, Kosuke, E-mail: morikako@protein.osaka-u.ac.jp},
abstractNote = {A stable stoichiometric complex of archaeal DNA polymerase with proliferating cell nuclear antigen (PCNA) was formed using a PCNA monomer mutant and the complex was successfully crystallized. 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 P2{sub 1}2{sub 1}2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 Å.},
doi = {10.1107/S1744309106004362},
journal = {Acta Crystallographica. Section F},
number = Pt 3,
volume = 62,
place = {United Kingdom},
year = {Wed Mar 01 00:00:00 EST 2006},
month = {Wed Mar 01 00:00:00 EST 2006}
}