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.
Replicative DNA polymerase interacts with processivity factors, the beta-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 A resolution using synchrotron radiation. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 A.
Nishida
Hirokazu
H
Department of Structural Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
Matsumiya
Shigeki
S
Tsuchiya
Daisuke
D
Ishino
Yoshizumi
Y
Morikawa
Kosuke
K
D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry*; D020027:Pyrococcus furiosus / Q000737:chemistry*
D018809:Proliferating Cell Nuclear Antigen; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase