RESUMEN
The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following: This study was in part supported by the Swiss National Foundation Grant No.: 31003A-156023 to Alessandro Sartori.
RESUMEN
Interstrand cross-link (ICL) hypersensitivity is a characteristic trait of Fanconi anemia (FA). Although FANCD2-associated nuclease 1 (FAN1) contributes to ICL repair, FAN1 mutations predispose to karyomegalic interstitial nephritis (KIN) and cancer rather than to FA. Thus, the biological role of FAN1 remains unclear. Because fork stalling in FAN1-deficient cells causes chromosomal instability, we reasoned that the key function of FAN1 might lie in the processing of halted replication forks. Here, we show that FAN1 contains a previously-uncharacterized PCNA interacting peptide (PIP) motif that, together with its ubiquitin-binding zinc finger (UBZ) domain, helps recruit FAN1 to ubiquitylated PCNA accumulated at stalled forks. This prevents replication fork collapse and controls their progression. Furthermore, we show that FAN1 preserves replication fork integrity by a mechanism that is distinct from BRCA2-dependent homologous recombination. Thus, targeting FAN1 activities and its interaction with ubiquitylated PCNA may offer therapeutic opportunities for treatment of BRCA-deficient tumors.
Asunto(s)
Proteína BRCA2/metabolismo , Exodesoxirribonucleasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína BRCA2/genética , Línea Celular Tumoral , Reparación del ADN/genética , Reparación del ADN/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Endodesoxirribonucleasas , Exodesoxirribonucleasas/genética , Humanos , Enzimas Multifuncionales , Antígeno Nuclear de Célula en Proliferación/genética , Unión Proteica/genética , Unión Proteica/fisiología , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
RUVBL1 (RuvB-like1) and RUVBL2 (RuvB-like 2) are integral components of multisubunit protein complexes involved in processes ranging from cellular metabolism, transcription and chromatin remodeling to DNA repair. Here, we show that although RUVBL1 and RUVBL2 are known to form heterodimeric complexes in which they stabilize each other, the subunits separate during cytokinesis. In anaphase-to-telophase transition, RUVBL1 localizes to structures of the mitotic spindle apparatus, where it partially co-localizes with polo-like kinase 1 (PLK1). The ability of PLK1 to phosphorylate RUVBL1-but not RUVBL2-in vitro and their physical association in vivo suggest that this kinase differentially regulates the function of the RuvB-like proteins during mitosis. We further show that siRNA-mediated knock-down of RuvB-like proteins causes severe defects in chromosome alignment and segregation. In addition, we show that the ATPase activity of RUVBL1 is indispensable for cell proliferation. Our data thus demonstrate that RUVBL1 is essential for efficient mitosis and proliferation.
Asunto(s)
Proteínas Portadoras/metabolismo , Segregación Cromosómica/fisiología , Citocinesis/fisiología , ADN Helicasas/metabolismo , Mitosis/fisiología , ATPasas Asociadas con Actividades Celulares Diversas , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/genética , Reparación del ADN , Células HeLa , Humanos , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismo , Quinasa Tipo Polo 1RESUMEN
The mammalian antibody repertoire is shaped by somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) loci of B lymphocytes. SHM and CSR are triggered by non-canonical, error-prone processing of G/U mismatches generated by activation-induced deaminase (AID). In birds, AID does not trigger SHM, but it triggers Ig gene conversion (GC), a 'homeologous' recombination process involving the Ig variable region and proximal pseudogenes. Because recombination fidelity is controlled by the mismatch repair (MMR) system, we investigated whether MMR affects GC in the chicken B cell line DT40. We show here that Msh6(-/-) and Pms2(-/-) DT40 cells display cell cycle defects, including genomic re-replication. However, although IgVλ GC tracts in MMR-deficient cells were slightly longer than in normal cells, Ig GC frequency, donor choice or the number of mutations per sequence remained unaltered. The finding that the avian MMR system, unlike that of mammals, does not seem to contribute towards the processing of G/U mismatches in vitro could explain why MMR is unable to initiate Ig GC in this species, despite initiating SHM and CSR in mammalian cells. Moreover, as MMR does not counteract or govern Ig GC, we report a rare example of 'homeologous' recombination insensitive to MMR.
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Pollos/genética , Citidina Desaminasa/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/deficiencia , Conversión Génica , Inmunoglobulinas/genética , Uracilo/metabolismo , Animales , Apoptosis , Ciclo Celular , Línea Celular , Forma del Núcleo Celular , Proliferación Celular , Reparación de la Incompatibilidad de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Recombinación HomólogaRESUMEN
Cytotoxicity of cisplatin and mitomycin C (MMC) is ascribed largely to their ability to generate interstrand crosslinks (ICLs) in DNA, which block the progression of replication forks. The processing of ICLs requires the Fanconi anemia (FA) pathway, excision repair, and translesion DNA synthesis (TLS). It also requires homologous recombination (HR), which repairs double-strand breaks (DSBs) generated by cleavage of the blocked replication forks. Here we describe KIAA1018, an evolutionarily conserved protein that has an N-terminal ubiquitin-binding zinc finger (UBZ) and a C-terminal nuclease domain. KIAA1018 is a 5'-->3' exonuclease and a structure-specific endonuclease that preferentially incises 5' flaps. Like cells from FA patients, human cells depleted of KIAA1018 are sensitized to ICL-inducing agents and display chromosomal instability. The link of KIAA1018 to the FA pathway is further strengthened by its recruitment to DNA damage through interaction of its UBZ domain with monoubiquitylated FANCD2. We therefore propose to name KIAA1018 FANCD2-associated nuclease, FAN1.