Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing

• Published in: Nature communications Vol. 13; no. 1; pp. 7833 - 15
• Main Authors: Blair, Kerry, Tehseen, Muhammad, Raducanu, Vlad-Stefan, Shahid, Taha, Lancey, Claudia, Rashid, Fahad, Crehuet, Ramon, Hamdan, Samir M., De Biasio, Alfredo
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 20.12.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 101/28; 631/45/173; 631/535/1258/1259; 82/16; Binding sites; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA biosynthesis; DNA Ligase ATP - genetics; DNA Ligase ATP - metabolism; DNA polymerase; DNA Polymerase III - metabolism; DNA Replication; Endonuclease; FEN1 protein; Flap Endonucleases - metabolism; Fragments; Humanities and Social Sciences; Humans; Ligases - metabolism; multidisciplinary; N-Terminus; Okazaki fragments; Proliferating cell nuclear antigen; Proliferating Cell Nuclear Antigen - metabolism; Science; Science (multidisciplinary); Sealing; Sliding; Structural models; Substrates; Tool holders
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-35475-z

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIP N-term ) and DNA binding domain (PIP DBD ). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIP N-term is released from PCNA and only PIP DBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation. In this work, Blair and co-authors used cryo-EM and in vitro assays to show that human Ligase 1 (Lig1) and Flap Endonuclease 1 (FEN1) form a toolbelt with the sliding clamp PCNA coordinating the sealing of Okazaki fragments.