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Dimerization-dependent serine protease activity of FAM111A prevents replication fork stalling at topoisomerase 1
Sowmiya Palani1, Yuka Machida2,3, Julia R Alvey4
1Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, MN, USA.
Abstract:
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.
Insights
Family with sequence similarity 111A (FAM111A) is a dimerization-dependent protease. Its dimerization is crucial for substrate cleavage and DNA replication but not for autocleavage, revealing distinct functional roles.
Area of Science:
- Molecular Biology
- Structural Biology
- Enzymology
Background:
- Family with sequence similarity 111A (FAM111A) is a serine protease involved in DNA replication and antiviral defense.
- Mutations in FAM111A's catalytic domain lead to hyper-autocleavage and are linked to genetic disorders characterized by developmental defects.
- The molecular structure of the FAM111A serine protease domain (SPD) remains uncharacterized, hindering a full understanding of its function.
Purpose of the Study:
- To elucidate the molecular architecture of the FAM111A serine protease domain (SPD).
- To investigate the role of dimerization in FAM111A's proteolytic activity and cellular functions.
- To understand the mechanism of FAM111A's substrate cleavage and its distinction from autocleavage.
Main Methods:
- X-ray crystallography was employed to determine the structure of the FAM111A serine protease domain.
- Site-directed mutagenesis studies were conducted to probe the function of specific FAM111A residues and domains.
- In vitro proteolytic assays and cellular DNA replication assays were used to assess FAM111A activity.
Main Results:
- FAM111A functions as a dimerization-dependent protease with a narrow, recessed active site exhibiting chymotrypsin-like substrate specificity.
- X-ray structures reveal that FAM111A dimerizes through its N-terminal helix, initiating an activation cascade involving disorder-to-order transitions.
- Dimerization is essential for in vitro proteolytic activity and for facilitating DNA replication at DNA-protein crosslinks, but dispensable for autocleavage.
Conclusions:
- Dimerization is a critical regulatory mechanism for FAM111A's enzymatic activity and its role in DNA replication.
- The study highlights a functional divergence in dimerization dependency between FAM111A's substrate cleavage and its autocleavage.
- Understanding FAM111A's structure-function relationship provides insights into genetic disorders associated with its mutations.
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