Single-molecule characterization of SV40 replisome and novel factors: human FPC and Mcm10

Abstract The simian virus 40 (SV40) replisome only encodes for its helicase; large T-antigen (L-Tag), while relying on the host for the remaining proteins, making it an intriguing model system. Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its acti...

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Veröffentlicht in:Nucleic acids research 2024-08, Vol.52 (15), p.8880-8896
Hauptverfasser: Ouyang, Yujing, Al-Amodi, Amani, Tehseen, Muhammad, Alhudhali, Lubna, Shirbini, Afnan, Takahashi, Masateru, Raducanu, Vlad-Stefan, Yi, Gang, Danazumi, Ammar Usman, De Biasio, Alfredo, Hamdan, Samir M
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container_end_page 8896
container_issue 15
container_start_page 8880
container_title Nucleic acids research
container_volume 52
creator Ouyang, Yujing
Al-Amodi, Amani
Tehseen, Muhammad
Alhudhali, Lubna
Shirbini, Afnan
Takahashi, Masateru
Raducanu, Vlad-Stefan
Yi, Gang
Danazumi, Ammar Usman
De Biasio, Alfredo
Hamdan, Samir M
description Abstract The simian virus 40 (SV40) replisome only encodes for its helicase; large T-antigen (L-Tag), while relying on the host for the remaining proteins, making it an intriguing model system. Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its activities and the identification of new factors remain largely unexplored. Herein, we in vitro reconstituted the SV40 replisome activities at the single-molecule level, including DNA unwinding by L-Tag and the single-stranded DNA-binding protein Replication Protein A (RPA), primer extension by DNA polymerase δ, and their concerted leading-strand synthesis. We show that RPA stimulates the processivity of L-Tag without altering its rate and that DNA polymerase δ forms a stable complex with L-Tag during leading-strand synthesis. Furthermore, similar to human and budding yeast Cdc45–MCM–GINS helicase, L-Tag uses the fork protection complex (FPC) and the mini-chromosome maintenance protein 10 (Mcm10) during synthesis. Hereby, we demonstrate that FPC increases this rate, and both FPC and Mcm10 increase the processivity by stabilizing stalled replisomes and increasing their chances of restarting synthesis. The detailed kinetics and novel factors of the SV40 replisome establish it as a closer mimic of the host replisome and expand its application as a model replication system. Graphical Abstract Graphical Abstract
doi_str_mv 10.1093/nar/gkae565
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Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its activities and the identification of new factors remain largely unexplored. Herein, we in vitro reconstituted the SV40 replisome activities at the single-molecule level, including DNA unwinding by L-Tag and the single-stranded DNA-binding protein Replication Protein A (RPA), primer extension by DNA polymerase δ, and their concerted leading-strand synthesis. We show that RPA stimulates the processivity of L-Tag without altering its rate and that DNA polymerase δ forms a stable complex with L-Tag during leading-strand synthesis. Furthermore, similar to human and budding yeast Cdc45–MCM–GINS helicase, L-Tag uses the fork protection complex (FPC) and the mini-chromosome maintenance protein 10 (Mcm10) during synthesis. Hereby, we demonstrate that FPC increases this rate, and both FPC and Mcm10 increase the processivity by stabilizing stalled replisomes and increasing their chances of restarting synthesis. The detailed kinetics and novel factors of the SV40 replisome establish it as a closer mimic of the host replisome and expand its application as a model replication system. 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Hereby, we demonstrate that FPC increases this rate, and both FPC and Mcm10 increase the processivity by stabilizing stalled replisomes and increasing their chances of restarting synthesis. The detailed kinetics and novel factors of the SV40 replisome establish it as a closer mimic of the host replisome and expand its application as a model replication system. Graphical Abstract Graphical Abstract</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>38967018</pmid><doi>10.1093/nar/gkae565</doi><tpages>17</tpages><orcidid>https://orcid.org/0000-0001-5192-1852</orcidid><oa>free_for_read</oa></addata></record>
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subjects Antigens, Polyomavirus Transforming - genetics
Antigens, Polyomavirus Transforming - metabolism
DNA Helicases - genetics
DNA Helicases - metabolism
DNA Polymerase III - genetics
DNA Polymerase III - metabolism
DNA Replication
DNA, Single-Stranded - metabolism
DNA, Viral - genetics
DNA, Viral - metabolism
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
DNA-Directed DNA Polymerase
Genome Integrity, Repair and
Humans
Minichromosome Maintenance Proteins - genetics
Minichromosome Maintenance Proteins - metabolism
Multienzyme Complexes
Replication Protein A - metabolism
Simian virus 40 - genetics
Simian virus 40 - metabolism
Single Molecule Imaging
Virus Replication
title Single-molecule characterization of SV40 replisome and novel factors: human FPC and Mcm10
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