RESUMEN
Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.
Asunto(s)
Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas Adaptadoras Transductoras de Señales , Proteína BRCA1/antagonistas & inhibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Humanos , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Proteínas Mad2/antagonistas & inhibidores , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión a Telómeros/antagonistas & inhibidores , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/antagonistas & inhibidores , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The Escherichia coli TetR-related transcriptional regulator RutR is involved in the coordination of pyrimidine and purine metabolism. Here we report that lysine acetylation modulates RutR function. Applying the genetic code expansion concept, we produced site-specifically lysine-acetylated RutR proteins. The crystal structure of lysine-acetylated RutR reveals how acetylation switches off RutR-DNA-binding. We apply the genetic code expansion concept in E. coli in vivo revealing the consequences of RutR acetylation on the transcriptional level. We propose a model in which RutR acetylation follows different kinetic profiles either reacting non-enzymatically with acetyl-phosphate or enzymatically catalysed by the lysine acetyltransferases PatZ/YfiQ and YiaC. The NAD+-dependent sirtuin deacetylase CobB reverses enzymatic and non-enzymatic acetylation of RutR playing a dual regulatory and detoxifying role. By detecting cellular acetyl-CoA, NAD+ and acetyl-phosphate, bacteria apply lysine acetylation of transcriptional regulators to sense the cellular metabolic state directly adjusting gene expression to changing environmental conditions.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lisina/metabolismo , Acetilación , NAD/metabolismo , Expresión Génica , Fosfatos/metabolismoRESUMEN
Lysine acetylation is a post-translational modification that is conserved from bacteria to humans. It is catalysed by the activities of lysine acetyltransferases, which use acetyl-CoA as the acetyl-donor molecule, and lysine deacetylases, which remove the acetyl moiety. Recently, it was reported that YcgC represents a new prokaryotic deacetylase family with no apparent homologies to existing deacetylases (Tu et al., 2015). Here we report the results of experiments which demonstrate that YcgC is not a deacetylase.