RESUMO
Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3, are predisposed to breast, ovarian and prostate cancers1-10 and the cancer-prone syndrome Fanconi anaemia11-13. The BRCA2 tumour suppressor protein-the product of BRCA2-is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects14,15, but the precise molecular roles of RAD51 paralogues in fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B-RAD51C-RAD51D-XRCC2 complex (BCDX2), revealing that RAD51C-RAD51D-XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments-which are essential for recombinational DNA repair-in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance.
Assuntos
Microscopia Crioeletrônica , Proteínas de Ligação a DNA , Complexos Multiproteicos , Rad51 Recombinase , Proteínas Supressoras de Tumor , Humanos , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Recombinação Homóloga , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Rad51 Recombinase/ultraestrutura , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/ultraestrutura , Inibidores de Poli(ADP-Ribose) Polimerases , Neoplasias/genética , Neoplasias/prevenção & controle , Proteômica , Simulação por Computador , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Quebras de DNA de Cadeia DuplaRESUMO
MutSα and MutSß play important roles in DNA mismatch repair and are linked to inheritable cancers and degenerative disorders. Here, we show that MSH2 and MSH3, the two components of MutSß, bind SLX4 protein, a scaffold for the assembly of the SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX) trinuclease complex. SMX promotes the resolution of Holliday junctions (HJs), which are intermediates in homologous recombinational repair. We find that MutSß binds HJs and stimulates their resolution by SLX1-SLX4 or SMX in reactions dependent upon direct interactions between MutSß and SLX4. In contrast, MutSα does not stimulate HJ resolution. MSH3-depleted cells exhibit reduced sister chromatid exchanges and elevated levels of homologous recombination ultrafine bridges (HR-UFBs) at mitosis, consistent with defects in the processing of recombination intermediates. These results demonstrate a role for MutSß in addition to its established role in the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease.
Assuntos
Resolvases de Junção Holliday/metabolismo , Proteínas MutS/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Instabilidade Genômica , Células HEK293 , Resolvases de Junção Holliday/fisiologia , Humanos , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS/metabolismo , Ligação Proteica , Recombinases/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiaeRESUMO
The protein biosynthetic machinery, composed of ribosomes, chaperones, and localization factors, is increasingly found to interact directly with factors dedicated to protein degradation. The coupling of these two opposing processes facilitates quality control of nascent polypeptides at each stage of their maturation. Sequential checkpoints maximize the overall fidelity of protein maturation, minimize the exposure of defective products to the bulk cellular environment, and protect organisms from protein misfolding diseases.
Assuntos
Biossíntese de Proteínas , Animais , Humanos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Transporte Proteico , Proteólise , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
Protein ubiquitination is catalyzed by ubiquitin-conjugating enzymes (E2s) in collaboration with ubiquitin-protein ligases (E3s). This process depends on nucleophilic attack by a substrate lysine on a thioester bond linking the C terminus of ubiquitin to a cysteine in the E2 active site. Different E2 family members display specificity for lysines in distinct contexts. We addressed the mechanistic basis for this lysine selectivity in Ubc1, an E2 that catalyzes the ubiquitination of lysine 48 (K48) in ubiquitin, leading to the formation of K48-linked polyubiquitin chains. We identified a cluster of polar residues near the Ubc1 active site, as well as a residue in ubiquitin itself, that are required for catalysis of K48-specific ubiquitin ligation, but not for general activity toward other lysines. Our results suggest that the active site of Ubc1, as well as the surface of ubiquitin, contains specificity determinants that channel specific lysines to the central residues involved directly in catalysis.