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

Su Hyung Park,Seong-Jung Kim,Kyoo-Young Lee,Kyungjae Myung

doi:10.1038/s41598-021-01336-w

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.

Highlights

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
We found that the protein levels of all replication factor C (RFC)/RFC-like complexes (RLCs) proteins in the pellet fraction were reduced in the Lap2α knockout cells (Fig. 6A)
We detected RFC1 and ATAD5 only in the nuclease- and high saltresistant fraction, while small RFC proteins were located in both soluble and pellet fractions (Fig. 1C and Supplementary Fig. 1), which were independent of cell cycle and DNA damage (Figs. 2, 3)

Summary

Introduction

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 A-type lamins (lamin A and C, lamin A/C) and B-type lamins, in a complex with lamina-associated polypeptide (LAP)-Emerin-MAN1 (LEM) domain proteins and lamin B receptor of the INM, respectively, tether heterochromatic genomic regions to the nuclear periphery[3]. These structures shape the spatial architecture of chromatin, which is closely related to gene regulation and replication timing in the interphase n ucleus[1,4,5]. RAD17-RLC loads another ring-shaped clamp, RAD9-RAD1-HUS1 (the 9-1-1 complex, Ddc1-Rad17‐Mec[3], in budding yeast)[22], which plays a role in checkpoint activation[23,24]

Methods

Results

Conclusion