RESUMEN
The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ultimately lead to diseases including cancer. Non-homologous end joining (NHEJ) is one of two core mechanisms utilized to repair DSBs. DNA-PK is a key component in this process and has recently been shown to form alternate long-range synaptic dimers. This has led to the proposal that these complexes can be formed before transitioning to a short-range synaptic complex. Here we present cryo-EM data representing an NHEJ supercomplex consisting of a trimer of DNA-PK in complex with XLF, XRCC4, and DNA Ligase IV. This trimer represents a complex of both long-range synaptic dimers. We discuss the potential role of the trimeric structure, and possible higher order oligomers, as structural intermediates in the NHEJ mechanism, or as functional DNA repair centers.
Asunto(s)
Enzimas Reparadoras del ADN , Reparación del ADN , Humanos , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Microscopía por Crioelectrón , Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP) , Proteína Quinasa Activada por ADN/metabolismo , ADN/genéticaRESUMEN
Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.
Asunto(s)
ADN Ligasa (ATP)/ultraestructura , Enzimas Reparadoras del ADN/ultraestructura , Proteína Quinasa Activada por ADN/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Complejos Multiproteicos/ultraestructura , Apoptosis/genética , Microscopía por Crioelectrón , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/genética , ADN Ligasa (ATP)/genética , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/ultraestructura , Complejos Multiproteicos/genética , Fosforilación/genéticaRESUMEN
Non-homologous end joining (NHEJ) is the preferred pathway for the repair of DNA double-strand breaks in humans. Here we describe three structural aspects of the repair pathway: stages, scaffolds and strings. We discuss the orchestration of DNA repair to guarantee robust and efficient NHEJ. We focus on structural studies over the past two decades, not only using X-ray diffraction, but also increasingly exploiting cryo-EM to investigate the macromolecular assemblies.
