Functional roles for FEN1 phosphate steering residues in multi-step substrate verification prior to reaction

Flap endonuclease 1 (FEN1) removes 5′-flaps from double-flap DNA junction intermediates during replication and repair. Substrate recognition and reaction site selection depend on two intrinsically disordered regions: the α4–α5 helical arch, through which the 5′-flap threads prior to catalysis, and the adjacent 3′-flap binding pocket, which allosterically signals disorder-to-order transition of the arch upon sensing a 3′-flap, committing the enzyme–DNA complex towards reaction. The phosphate steering hypothesis proposes that conserved, positively charged amino acids in α4/α5 facilitate passage of 5′-flap DNA through the arch during threading and position the target phosphate diester for hydrolysis; however, supporting evidence is limited and mechanistic details are currently lacking. We investigated functional roles for these residues using kinetic and spectroscopic methods, finding that alanine substitutions of Arg103, Arg104, Arg129 and Lys132 modestly reduce the catalytic rate and the stability of 5′-flap threading. Following arch ordering, distortion of the reacting DNA duplex is necessary for active site transfer of the target cut site, and we identified key roles in this process for two substrate-facing residues from α5, Lys125 and Arg129. Concurrently, ‘back-of-arch’ residues Arg104 and Lys132 contact the +1 phosphate to precisely position the target phosphodiester for hydrolysis. Helicity-disrupting mutations in α4/α5, designed to impair ordering, reduced the catalytic rate and severely inhibited allosteric signalling of 3′-flap recognition to the active site. These findings define critical functional roles for phosphate steering residues in the FEN1 mechanism, and inform a deeper understanding of how coordinated substrate verification optimises targeting specificity to preserve genome integrity.