RESUMEN
Efficient and accurate DNA synthesis is enabled by DNA polymerase fidelity checkpoints that promote insertion of the right instead of wrong nucleotide. Erroneous X-family polymerase (pol) λ nucleotide insertion leads to genomic instability in double strand break and base-excision repair. Here, time-lapse crystallography captures intermediate catalytic states of pol λ undergoing right and wrong natural nucleotide insertion. The revealed nucleotide sensing mechanism responds to base pair geometry through active site deformation to regulate global polymerase-substrate complex alignment in support of distinct optimal (right) or suboptimal (wrong) reaction pathways. An induced fit during wrong but not right insertion, and associated metal, substrate, side chain and pyrophosphate reaction dynamics modulated nucleotide insertion. A third active site metal hastened right but not wrong insertion and was not essential for DNA synthesis. The previously hidden fidelity checkpoints uncovered reveal fundamental strategies of polymerase DNA repair synthesis in genomic instability.
Asunto(s)
ADN Polimerasa beta , Nucleótidos , ADN/metabolismo , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Inestabilidad Genómica , Humanos , Cinética , Modelos Moleculares , Nucleótidos/metabolismoRESUMEN
Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase µ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Mutagénesis/genética , Nucleótidos/metabolismo , Adenina/metabolismo , Emparejamiento Base , Biocatálisis , Dominio Catalítico , Citosina/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Humanos , Modelos Moleculares , Mutagénesis Insercional/genética , Oxidación-ReducciónRESUMEN
Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) µ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol µ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol µ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol µ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Ribonucleótidos/metabolismo , Adenina/metabolismo , Calcio/metabolismo , Dominio Catalítico , Citosina/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiguanina/química , Nucleótidos de Desoxiguanina/metabolismo , Humanos , Cinética , Manganeso/metabolismo , Modelos Moleculares , Oxidación-ReducciónRESUMEN
DNA polymerase (pol) µ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol µ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PPi. The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5'-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol µ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.DNA polymerase (pol) µ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.