RESUMEN
DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognize opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3'-terminus of a primer strand, which is recognized by breaking the terminal base pair to generate a substrate with a single nucleotide 3'-flap. This recognition event allosterically signals hydrolytic removal of the 5'-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved 'wedge' residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated 'loop-wedge' mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognize irregular DNA structures. These new findings reveal how FEN1 precisely couples 3'-flap verification to function.
Asunto(s)
Reparación del ADN , Replicación del ADN , ADN/genética , Endonucleasas de ADN Solapado/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Dominio Catalítico , ADN/química , ADN/metabolismo , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/metabolismo , Humanos , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
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'-terminiin 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 Tyr(40), Asp(181), and Arg(100)and a reacting duplex 5'-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5'-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage.
Asunto(s)
ADN/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Dicroismo Circular , ADN/química , Reparación del ADN , Transferencia Resonante de Energía de Fluorescencia , Humanos , Conformación de Ácido Nucleico , Especificidad por SustratoRESUMEN
During efforts to prepare the known compound , a new tetracyclic compound, called VG1, was prepared in six steps. This compound was found to have good activity as an inhibitor of nonsense-mediated mRNA decay.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Degradación de ARNm Mediada por Codón sin Sentido/efectos de los fármacos , ARN Mensajero/metabolismo , Relación Dosis-Respuesta a Droga , Compuestos Heterocíclicos de 4 o más Anillos/química , Estructura MolecularRESUMEN
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.
Asunto(s)
ADN/genética , Endonucleasas de ADN Solapado/metabolismo , Inestabilidad Genómica , Fosfatos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , ADN/química , ADN/metabolismo , Reparación del ADN , Replicación del ADN , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/genética , Humanos , Mutación , Fosfatos/química , Alineación de Secuencia , Especificidad por SustratoRESUMEN
[This corrects the article DOI: 10.1038/ncomms15855.].