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

• Published in: Nucleic acids research Vol. 52; no. 15; pp. 8880 - 8896
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 27.08.2024
• 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
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkae565

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