DNA Replication and Repair

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In our previous study, we have solved X-ray crystal structures of the FAM111A serine protease domain (SPD). Our biochemical and structural studies suggested that FAM111A SPD forms a homodimer through the alpha-1 helix at the N-terminus, where two subunits are held together through hydrophobic interactions. Substitution of the valine residue on the dimerization interface with aspartate diminished dimer formation and protease activity of the SPD in vitro, as well as the ability to protect replication forks from DPCs induced by a topoisomerase inhibitor, camptothecin, in cells. To understand how dimerization of the SPD promotes the protease activity, we have solved X-ray crystal structures of an SPD mutant that lacks a part of the dimerization interface. Interestingly, not only the dimerization interface, but also the oxyanion hole, an active site structure critical for protease activity, is disordered in the monomeric mutants. These new data reveal a mechanism of the dimerization-dependent activity of the FAM111A SPD, in which dimerization triggers a cascade of disorder-to-order transitions that leads to the stabilization of the oxyanion hole. These structural studies will help us determine potential mechanisms that regulate the protease activity of FAM111A at replication forks stalled at DPCs and facilitate the development of FAM111A inhibitors as a sensitizers of anti-cancer therapeutics including topoisomerase inhibitors.