DNA Replication and Repair

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA replication and threaten genome stability. The serine protease FAM111A plays a key role at replication forks by promoting DNA replication through the sites of DPCs. In addition to its role in genome maintenance, FAM111A also functions as an antiviral factor, though the mechanisms by which viruses evade its activity remain poorly understood. Our research aims to better understand the molecular functions of FAM111A and determine how it contributes to both DNA repair and the cellular response to viral infection. In this project, we investigated the role of the rabbitpox virus (RPXV) serpin SPI-1, which is required for viral replication in restrictive host cells. Previous studies suggested that SPI-1 promotes infection by antagonizing FAM111A, but direct evidence of this interaction was lacking. In this funding period, we demonstrated that SPI-1 directly inhibits the protease activity of FAM111A through covalent complex formation, which is a hallmark of the serpin inhibition mechanism. In vitro assays showed that SPI-1 exhibits high specificity for FAM111A over other serine proteases. Mutagenesis study of SPI-1's reactive center loop (RCL) revealed key residues required for complex formation and FAM111A inhibition. These mutations resulted in a range of defects in supporting RPXV replication in non-permissive cells, which closely correlated with their biochemical activity against FAM111A. Altogether, our findings establish FAM111A as an antiviral factor and identify SPI-1 as a viral inhibitor that neutralizes FAM111A to enable infection. This work enhances our understanding of host-virus interactions and supports our long-term goal of defining the role of FAM111A in cellular response to replication stress and viral infection.