Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy
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RESEARCH ARTICLE
Open Access
Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy Kouta Mayanagi1*†, Keisuke Oki2†, Naoyuki Miyazaki3,4, Sonoko Ishino2, Takeshi Yamagami2, Kosuke Morikawa5, Kenji Iwasaki3,4, Daisuke Kohda1, Tsuyoshi Shirai6* and Yoshizumi Ishino2*
Abstract Background: DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3′-5′ editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM). Results: Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a β-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro. Conclusions: Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life. Keywords: Archaea, Replisome, PolD, PCNA, Processive DNA synthesis
* Correspondence: [email protected]; [email protected]; [email protected] † Kouta Mayanagi and Keisuke Oki contributed equally to this work. 1 Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka-shi, Fukuoka 812-8582, Japan 6 Department of Bioscience, Nagahama Institute of Bio-Science and Technology, Tamura 1266, Nagahama, Shiga 526-0829, Japan 2 Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, Fukuoka 819-0395, Japan Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 Internat
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