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Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism
Proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity clamp for eukaryotic DNA polymerases and a binding platform for many DNA replication and repair proteins. The enzymatic activities of PCNA loading and unloading have been studied extensively in vitro. However, the subc...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8575788/ https://www.ncbi.nlm.nih.gov/pubmed/34751190 http://dx.doi.org/10.1038/s41598-021-01336-w |
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author | Park, Su Hyung Kim, Seong-jung Myung, Kyungjae Lee, Kyoo-young |
author_facet | Park, Su Hyung Kim, Seong-jung Myung, Kyungjae Lee, Kyoo-young |
author_sort | Park, Su Hyung |
collection | PubMed |
description | Proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity clamp for eukaryotic DNA polymerases and a binding platform for many DNA replication and repair proteins. The enzymatic activities of PCNA loading and unloading have been studied extensively in vitro. However, the subcellular locations of PCNA loaders, replication complex C (RFC) and CTF18-RFC-like-complex (RLC), and PCNA unloader ATAD5-RLC remain elusive, and the role of their subunits RFC2-5 is unknown. Here we used protein fractionation to determine the subcellular localization of RFC and RLCs and affinity purification to find molecular requirements for the newly defined location. All RFC/RLC proteins were detected in the nuclease-resistant pellet fraction. RFC1 and ATAD5 were not detected in the non-ionic detergent-soluble and nuclease-susceptible chromatin fractions, independent of cell cycle or exogenous DNA damage. We found that small RFC proteins contribute to maintaining protein levels of the RFC/RLCs. RFC1, ATAD5, and RFC4 co-immunoprecipitated with lamina-associated polypeptide 2 (LAP2) α which regulates intranuclear lamin A/C. LAP2α knockout consistently reduced detection of RFC/RLCs in the pellet fraction, while marginally affecting total protein levels. Our findings strongly suggest that PCNA-mediated DNA transaction occurs through regulatory machinery associated with nuclear structures, such as the nuclear matrix. |
format | Online Article Text |
id | pubmed-8575788 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-85757882021-11-09 Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism Park, Su Hyung Kim, Seong-jung Myung, Kyungjae Lee, Kyoo-young Sci Rep Article Proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity clamp for eukaryotic DNA polymerases and a binding platform for many DNA replication and repair proteins. The enzymatic activities of PCNA loading and unloading have been studied extensively in vitro. However, the subcellular locations of PCNA loaders, replication complex C (RFC) and CTF18-RFC-like-complex (RLC), and PCNA unloader ATAD5-RLC remain elusive, and the role of their subunits RFC2-5 is unknown. Here we used protein fractionation to determine the subcellular localization of RFC and RLCs and affinity purification to find molecular requirements for the newly defined location. All RFC/RLC proteins were detected in the nuclease-resistant pellet fraction. RFC1 and ATAD5 were not detected in the non-ionic detergent-soluble and nuclease-susceptible chromatin fractions, independent of cell cycle or exogenous DNA damage. We found that small RFC proteins contribute to maintaining protein levels of the RFC/RLCs. RFC1, ATAD5, and RFC4 co-immunoprecipitated with lamina-associated polypeptide 2 (LAP2) α which regulates intranuclear lamin A/C. LAP2α knockout consistently reduced detection of RFC/RLCs in the pellet fraction, while marginally affecting total protein levels. Our findings strongly suggest that PCNA-mediated DNA transaction occurs through regulatory machinery associated with nuclear structures, such as the nuclear matrix. Nature Publishing Group UK 2021-11-08 /pmc/articles/PMC8575788/ /pubmed/34751190 http://dx.doi.org/10.1038/s41598-021-01336-w Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Park, Su Hyung Kim, Seong-jung Myung, Kyungjae Lee, Kyoo-young Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism |
title | Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism |
title_full | Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism |
title_fullStr | Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism |
title_full_unstemmed | Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism |
title_short | Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism |
title_sort | characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8575788/ https://www.ncbi.nlm.nih.gov/pubmed/34751190 http://dx.doi.org/10.1038/s41598-021-01336-w |
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