RESUMO
During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteína do Grupo de Complementação L da Anemia de Fanconi , Recombinação Homóloga , Enzimas de Conjugação de Ubiquitina , Humanos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Sistemas CRISPR-Cas , Ubiquitinação , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Células HEK293 , Reparo de DNA por Recombinação , Reparo do DNA , Reparo do DNA por Junção de Extremidades , DNA HelicasesRESUMO
Homologous Recombination (HR) is a high-fidelity repair mechanism of DNA Double-Strand Breaks (DSBs), which are induced by irradiation, genotoxic chemicals or physiological DNA damaging processes. DSBs are also generated as intermediates during the repair of interstrand crosslinks (ICLs). In this context, the Fanconi anemia (FA) core complex, which is effectively recruited to ICLs, promotes HR-mediated DSB-repair. However, whether the FA core complex also promotes HR at ICL-independent DSBs remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen with cells carrying the DSB-repair reporter DSB-Spectrum. Using isogenic cell-line models, we validated the HR-function of FANCL and Ube2T, and demonstrated a similar function for their ubiquitination-substrate FANCD2. We further show that FANCL and Ube2T are directly recruited to DSBs and are required for the accumulation of FANCD2 at these break sites. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of the nuclease CtIP at DSBs, and consequently for optimal end-resection and Rad51 loading. CtIP overexpression rescues HR in FANCL-deficient cells, validating that FANCL primarily regulates HR by promoting CtIP recruitment. Together, these data demonstrate that the FA core complex and FANCD2 have a dual genome maintenance function by promoting repair of DSBs as well as the repair of ICLs.
RESUMO
RPA has been shown to protect single-stranded DNA (ssDNA) intermediates from instability and breakage. RPA binds ssDNA with sub-nanomolar affinity, yet dynamic turnover is required for downstream ssDNA transactions. How ultrahigh-affinity binding and dynamic turnover are achieved simultaneously is not well understood. Here we reveal that RPA has a strong propensity to assemble into dynamic condensates. In solution, purified RPA phase separates into liquid droplets with fusion and surface wetting behavior. Phase separation is stimulated by sub-stoichiometric amounts of ssDNA, but not RNA or double-stranded DNA, and ssDNA gets selectively enriched in RPA condensates. We find the RPA2 subunit required for condensation and multi-site phosphorylation of the RPA2 N-terminal intrinsically disordered region to regulate RPA self-interaction. Functionally, quantitative proximity proteomics links RPA condensation to telomere clustering and integrity in cancer cells. Collectively, our results suggest that RPA-coated ssDNA is contained in dynamic RPA condensates whose properties are important for genome organization and stability.