Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy
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 cata...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-10-01
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| Series: | BMC Biology |
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| Online Access: | http://link.springer.com/article/10.1186/s12915-020-00889-y |
| _version_ | 1818504102584254464 |
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| author | Kouta Mayanagi Keisuke Oki Naoyuki Miyazaki Sonoko Ishino Takeshi Yamagami Kosuke Morikawa Kenji Iwasaki Daisuke Kohda Tsuyoshi Shirai Yoshizumi Ishino |
| author_facet | Kouta Mayanagi Keisuke Oki Naoyuki Miyazaki Sonoko Ishino Takeshi Yamagami Kosuke Morikawa Kenji Iwasaki Daisuke Kohda Tsuyoshi Shirai Yoshizumi Ishino |
| author_sort | Kouta Mayanagi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-10T21:32:34Z |
| format | Article |
| id | doaj.art-d2e48c69b22042d58ea2b5bf273cd9c2 |
| institution | Directory Open Access Journal |
| issn | 1741-7007 |
| language | English |
| last_indexed | 2024-12-10T21:32:34Z |
| publishDate | 2020-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biology |
| spelling | doaj.art-d2e48c69b22042d58ea2b5bf273cd9c22022-12-22T01:32:45ZengBMCBMC Biology1741-70072020-10-0118112010.1186/s12915-020-00889-yTwo conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopyKouta Mayanagi0Keisuke Oki1Naoyuki Miyazaki2Sonoko Ishino3Takeshi Yamagami4Kosuke Morikawa5Kenji Iwasaki6Daisuke Kohda7Tsuyoshi Shirai8Yoshizumi Ishino9Medical Institute of Bioregulation, Kyushu UniversityDepartment of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu UniversityInstitute for Protein Research, Osaka UniversityDepartment of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu UniversityDepartment of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu UniversityDepartment of Gene Mechanisms, Graduate School of Biostudies, Kyoto UniversityInstitute for Protein Research, Osaka UniversityMedical Institute of Bioregulation, Kyushu UniversityDepartment of Bioscience, Nagahama Institute of Bio-Science and TechnologyDepartment of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu UniversityAbstract 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.http://link.springer.com/article/10.1186/s12915-020-00889-yArchaeaReplisomePolDPCNAProcessive DNA synthesis |
| spellingShingle | Kouta Mayanagi Keisuke Oki Naoyuki Miyazaki Sonoko Ishino Takeshi Yamagami Kosuke Morikawa Kenji Iwasaki Daisuke Kohda Tsuyoshi Shirai Yoshizumi Ishino Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy BMC Biology Archaea Replisome PolD PCNA Processive DNA synthesis |
| title | Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy |
| title_full | Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy |
| title_fullStr | Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy |
| title_full_unstemmed | Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy |
| title_short | Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy |
| title_sort | two conformations of dna polymerase d pcna dna an archaeal replisome complex revealed by cryo electron microscopy |
| topic | Archaea Replisome PolD PCNA Processive DNA synthesis |
| url | http://link.springer.com/article/10.1186/s12915-020-00889-y |
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