Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex.
DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the three-dimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNA-interacting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.
Medical Institute of Bioregulation, Kyushu University and Institute for Bioinformatics Research and Development, Japan Science and Technology Agency, 3-1-1 Maidashi, Higashi-ku, Fukuoka-shi, Fukuoka 812-8582, Japan. maya@bioreg.kyushu-u.ac.jp
Kouta
K
Mayanagi
Shinichi
S
Kiyonari
Hirokazu
H
Nishida
Mihoko
M
Saito
Daisuke
D
Kohda
Yoshizumi
Y
Ishino
Tsuyoshi
T
Shirai
Kosuke
K
Morikawa
D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D008854:Microscopy, Electron; D008958:Models, Molecular; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D019711:Pyrococcus / Q000201:enzymology; D019809:Streptavidin / Q000378:metabolism
D018809:Proliferating Cell Nuclear Antigen; D018698:Glutamic Acid; D004247:DNA; D019809:Streptavidin; D019951:DNA Polymerase beta
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