Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism

Abstract 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,...

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Autores principales:	Su Hyung Park, Seong-jung Kim, Kyungjae Myung, Kyoo-young Lee
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3cdf03e11e53435abfa52199b12d0460
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spelling	oai:doaj.org-article:3cdf03e11e53435abfa52199b12d04602021-11-14T12:20:31ZCharacterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism10.1038/s41598-021-01336-w2045-2322https://doaj.org/article/3cdf03e11e53435abfa52199b12d04602021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-01336-whttps://doaj.org/toc/2045-2322Abstract 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.Su Hyung ParkSeong-jung KimKyungjae MyungKyoo-young LeeNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-16 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Su Hyung Park Seong-jung Kim Kyungjae Myung Kyoo-young Lee Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism
description	Abstract 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	article
author	Su Hyung Park Seong-jung Kim Kyungjae Myung Kyoo-young Lee
author_facet	Su Hyung Park Seong-jung Kim Kyungjae Myung Kyoo-young Lee
author_sort	Su Hyung Park
title	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_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_sort	characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/3cdf03e11e53435abfa52199b12d0460
work_keys_str_mv	AT suhyungpark characterizationofsubcellularlocalizationofeukaryoticclamploaderunloaderanditsregulatorymechanism AT seongjungkim characterizationofsubcellularlocalizationofeukaryoticclamploaderunloaderanditsregulatorymechanism AT kyungjaemyung characterizationofsubcellularlocalizationofeukaryoticclamploaderunloaderanditsregulatorymechanism AT kyooyounglee characterizationofsubcellularlocalizationofeukaryoticclamploaderunloaderanditsregulatorymechanism
_version_	1718429281109934080

Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism

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