RESUMO
The chromatin-binding protein 53BP1 promotes DNA repair by orchestrating the recruitment of downstream effectors including PTIP, RIF1, and shieldin to DNA double-strand break sites. While we know how PTIP recognizes 53BP1, the molecular details of RIF1 recruitment to DNA-damage sites remains undefined. Here, we report that RIF1 is a phosphopeptide-binding protein that directly interacts with three phosphorylated 53BP1 epitopes. The RIF1-binding sites on 53BP1 share an essential LxL motif followed by two closely apposed phosphorylated residues. Simultaneous mutation of these sites on 53BP1 abrogates RIF1 accumulation into ionizing-radiation-induced foci, but surprisingly, only fully compromises 53BP1-dependent DNA repair when an alternative mode of shieldin recruitment to DNA-damage sites is also disabled. Intriguingly, this alternative mode of recruitment still depends on RIF1 but does not require its interaction with 53BP1. RIF1 therefore employs phosphopeptide recognition to promote DNA repair but also modifies shieldin action independently of 53BP1 binding.
Assuntos
Fosfopeptídeos , Proteínas de Ligação a Telômeros , Proteína BRCA1/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Fosfopeptídeos/genética , Fosfopeptídeos/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Neoplasias Mamárias Experimentais/enzimologia , Animais , Proteína BRCA1/genética , DNA Helicases/deficiência , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Retroalimentação Fisiológica , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Interferência de RNA , RecQ Helicases/genética , RecQ Helicases/metabolismo , Fase S , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor/genéticaRESUMO
Mitotic cells inactivate DNA double-strand break (DSB) repair, but the rationale behind this suppression remains unknown. Here, we unravel how mitosis blocks DSB repair and determine the consequences of repair reactivation. Mitotic kinases phosphorylate the E3 ubiquitin ligase RNF8 and the nonhomologous end joining factor 53BP1 to inhibit their recruitment to DSB-flanking chromatin. Restoration of RNF8 and 53BP1 accumulation at mitotic DSB sites activates DNA repair but is, paradoxically, deleterious. Aberrantly controlled mitotic DSB repair leads to Aurora B kinase-dependent sister telomere fusions that produce dicentric chromosomes and aneuploidy, especially in the presence of exogenous genotoxic stress. We conclude that the capacity of mitotic DSB repair to destabilize the genome explains the necessity for its suppression during mitosis, principally due to the fusogenic potential of mitotic telomeres.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Mitose/fisiologia , Homeostase do Telômero/fisiologia , Telômero/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , Telômero/genética , Homeostase do Telômero/genética , Transativadores/genética , Transativadores/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone 'code' produced by DSB signalling.
Assuntos
Dano ao DNA , Histonas/química , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.