Dimerization-dependent serine protease activity of FAM111A prevents replication fork stalling at topoisomerase 1 cleavage complexes

• Published in: Nature communications Vol. 15; no. 1; pp. 2064 - 16
• Main Authors: Palani, Sowmiya, Machida, Yuka, Alvey, Julia R., Mishra, Vandana, Welter, Allison L., Cui, Gaofeng, Bragantini , Benoît, Botuyan, Maria Victoria, Cong, Anh T. Q., Mer, Georges, Schellenberg, Matthew J., Machida, Yuichi J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 07.03.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/337/1427/1979; 631/45/173; 631/535/1266; 82/16; 82/80; 82/83; Antiviral drugs; BASIC BIOLOGICAL SCIENCES; Chymotrypsin; Cleavage; Crystal defects; Deoxyribonucleic acid; Dimerization; DNA; DNA adducts; DNA biosynthesis; DNA damage; DNA Replication; DNA topoisomerase; Enzyme mechanisms; Enzymes; Genetic disorders; Humanities and Social Sciences; Missense mutation; Molecular structure; multidisciplinary; Mutagenesis; Mutation; Peptides; Protease; Proteins; Proteolysis; Replication; Science; Science & Technology; Science (multidisciplinary); Serine; Serine Endopeptidases - metabolism; Serine Proteases; Serine proteinase; Stalling; Substrates; X-ray crystallography; Yeast
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-46207-w

FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme’s biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A’s function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage. FAM111A is a serine protease important for DNA replication and antiviral defense. Here, the authors report that the FAM111A dimerization is crucial for its proteolytic activity and for promoting DNA replication at trapped topoisomerase I.