DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1 Catalyzed Reaction.

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5'-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5'-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5'-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5'-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Y40, D181 and R100 and a reacting duplex 5'-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound, 5'-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage

Corrigendum: Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability.

[This corrects the article DOI: 10.1038/ncomms15855.]

Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability.

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via 'phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability

Corrigendum: Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability.

[This corrects the article DOI: 10.1038/ncomms15855.]