RESUMO
Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Here, we use single-molecule imaging to demonstrate that Rad54, a conserved Snf2-like protein found in all eukaryotes, switches the search from the diffusion-based pathways characteristic of the basal HR machinery to an active process in which DNA sequences are aligned via an ATP-dependent molecular motor-driven mechanism. We further demonstrate that Rad54 disrupts the donor template strands, enabling the search to take place within a migrating DNA bubble-like structure that is bound by replication protein A (RPA). Our results reveal that Rad54, working together with RPA, fundamentally alters how DNA sequences are aligned during HR.
Assuntos
Trifosfato de Adenosina/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , DNA/genética , Recombinação Homóloga/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Hidrólise , Saccharomyces cerevisiae/genética , Alinhamento de Sequência/métodosRESUMO
Mutations in the BRCA1 and BRCA2 genes predispose afflicted individuals to breast, ovarian, and other cancers. The BRCA-encoded products form complexes with other tumor suppressor proteins and with the recombinase enzyme RAD51 to mediate chromosome damage repair by homologous recombination and also to protect stressed DNA replication forks against spurious nucleolytic attrition. Understanding how the BRCA tumor suppressor network executes its biological functions would provide the foundation for developing targeted cancer therapeutics, but progress in this area has been greatly hampered by the challenge of obtaining purified BRCA complexes for mechanistic studies. In this article, we review how recent effort begins to overcome this technical challenge, leading to functional and structural insights into the biochemical attributes of these complexes and the multifaceted roles that they fulfill in genome maintenance. We also highlight the major mechanistic questions that remain.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Redes Reguladoras de Genes , Rad51 Recombinase/genética , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , DNA/química , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Feminino , Genoma Humano , Instabilidade Genômica , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The tumour suppressor breast cancer type 1 susceptibility protein (BRCA1) promotes DNA double-strand break (DSB) repair by homologous recombination and protects DNA replication forks from attrition. BRCA1 partners with BRCA1-associated RING domain protein 1 (BARD1) and other tumour suppressor proteins to mediate the initial nucleolytic resection of DNA lesions and the recruitment and regulation of the recombinase RAD51. The discovery of the opposing functions of BRCA1 and the p53-binding protein 1 (53BP1)-associated complex in DNA resection sheds light on how BRCA1 influences the choice of homologous recombination over non-homologous end joining and potentially other mutagenic pathways of DSB repair. Understanding the functional crosstalk between BRCA1-BARD1 and its cofactors and antagonists will illuminate the molecular basis of cancers that arise from a deficiency or misregulation of chromosome damage repair and replication fork maintenance. Such knowledge will also be valuable for understanding acquired tumour resistance to poly(ADP-ribose) polymerase (PARP) inhibitors and other therapeutics and for the development of new treatments. In this Review, we discuss recent advances in elucidating the mechanisms by which BRCA1-BARD1 functions in DNA repair, replication fork maintenance and tumour suppression, and its therapeutic relevance.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Proteínas Supressoras de Tumor/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Neoplasias da Mama/patologia , Reparo do DNA por Junção de Extremidades/genética , Replicação do DNA/genética , Feminino , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica/genética , Reparo de DNA por Recombinação/genéticaRESUMO
The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.
Assuntos
Dano ao DNA , Replicação do DNA , RecQ Helicases , Homeostase do Telômero , Telômero , RecQ Helicases/metabolismo , RecQ Helicases/genética , Humanos , Telômero/metabolismo , Telômero/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Linhagem Celular TumoralRESUMO
R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.
Assuntos
Estruturas R-Loop , Ribonucleases , Humanos , Estruturas R-Loop/genética , Ribonucleases/genética , RNA/genética , DNA , Replicação do DNA , DNA Helicases/genética , Ribonuclease H/genética , Ribonuclease H/metabolismo , Instabilidade GenômicaRESUMO
Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair single-strand DNA (ssDNA) with a homologous double-strand DNA (dsDNA) template. Here, we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a ninth nucleotide coincides with an additional reduction in binding free energy, and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination.
Assuntos
Recombinação Homóloga , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pareamento Cromossômico , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Alinhamento de SequênciaRESUMO
Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.
Assuntos
RNA Longo não Codificante , Homeostase do Telômero , Cromatina/genética , Proteômica , Telômero/genética , Telômero/metabolismo , RNA Longo não Codificante/genética , HomeostaseRESUMO
Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres1,2. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear3-5. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments (PICh) to capture the telomeric DNA damage response proteome during BITS1,6. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Recombinação Homóloga , Telômero , Moldes Genéticos , Animais , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Mamíferos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteômica , Rad51 Recombinase/metabolismo , Telômero/genética , Telômero/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Immune checkpoint blockade (ICB) immunotherapy is a first-line treatment for selected cancers, yet the mechanisms of its efficacy remain incompletely understood. Furthermore, only a minority of patients with cancer benefit from ICB, and there is a lack of fully informative treatment response biomarkers. Selectively exploiting defects in DNA damage repair is also a standard treatment for cancer, spurred by enhanced understanding of the DNA damage response (DDR). DDR and ICB are closely linked-faulty DDR produces immunogenic cancer neoantigens that can increase the efficacy of ICB therapy, and tumour mutational burden is a good but imperfect biomarker for the response to ICB. DDR studies in ICB efficacy initially focused on contributions to neoantigen burden. However, a growing body of evidence suggests that ICB efficacy is complicated by the immunogenic effects of nucleic acids generated from exogenous DNA damage or endogenous processes such as DNA replication. Chemotherapy, radiation, or selective DDR inhibitors (such as PARP inhibitors) can generate aberrant nucleic acids to induce tumour immunogenicity independently of neoantigens. Independent of their functions in immunity, targets of immunotherapy such as cyclic GMP-AMP synthase (cGAS) or PD-L1 can crosstalk with DDR or the DNA repair machinery to influence the response to DNA-damaging agents. Here we review the rapidly evolving, multifaceted interfaces between DDR, nucleic acid immunogenicity and immunotherapy efficacy, focusing on ICB. Understanding these interrelated processes could explain ICB treatment failures and reveal novel exploitable therapeutic vulnerabilities in cancers. We conclude by addressing major unanswered questions and new research directions.
Assuntos
Dano ao DNA , Inibidores de Checkpoint Imunológico , Imunoterapia , Neoplasias , Ácidos Nucleicos , Humanos , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Reparo do DNA , Imunoterapia/métodos , Imunoterapia/tendências , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Ácidos Nucleicos/metabolismo , Replicação do DNA , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Mutação , Biomarcadores Tumorais , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêuticoRESUMO
Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3-6 inactivate DNA binding and the HR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.
Assuntos
Proteínas de Ligação a DNA , Recombinação Homóloga , Complexos Multiproteicos , Humanos , Microscopia Crioeletrônica , Replicação do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Neoplasias/genética , Nucleoproteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Rad51 Recombinase/ultraestrutura , Especificidade por SubstratoRESUMO
Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.
Assuntos
Adenosina Trifosfatases/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Recombinação Homóloga , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Imagem Individual de Molécula , Proteína Vermelha FluorescenteRESUMO
Nonscheduled R loops represent a major source of DNA damage and replication stress. Cells have different ways to prevent R-loop accumulation. One mechanism relies on the conserved THO complex in association with cotranscriptional RNA processing factors including the RNA-dependent ATPase UAP56/DDX39B and histone modifiers such as the SIN3 deacetylase in humans. We investigated the function of UAP56/DDX39B in R-loop removal. We show that UAP56 depletion causes R-loop accumulation, R-loop-mediated genome instability, and replication fork stalling. We demonstrate an RNA-DNA helicase activity in UAP56 and show that its overexpression suppresses R loops and genome instability induced by depleting five different unrelated factors. UAP56/DDX39B localizes to active chromatin and prevents the accumulation of RNA-DNA hybrids over the entire genome. We propose that, in addition to its RNA processing role, UAP56/DDX39B is a key helicase required to eliminate harmful cotranscriptional RNA structures that otherwise would block transcription and replication.
Assuntos
RNA Helicases DEAD-box/metabolismo , Genoma/genética , Estruturas R-Loop/genética , Transcrição Gênica/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/genética , Expressão Gênica/genética , Instabilidade Genômica/genética , Humanos , Células K562RESUMO
The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas F-Box/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas F-Box/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Proteólise , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3' single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from â¼3-5 kb/h up to â¼10-20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection.
Assuntos
Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Cinética , Mutação , Domínios Proteicos , Transporte Proteico , Proteína Rad52 de Recombinação e Reparo de DNA/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
MRE11 nuclease forms a trimeric complex (MRN) with RAD50 and NBS1 and plays a central role in preventing genomic instability. When DNA double-strand breaks (DSBs) occur, MRN is quickly recruited to the damage site and initiates DNA end resection; accordingly, MRE11 must be tightly regulated to avoid inefficient repair or nonspecific resection. Here, we show that MRE11 and RAD50 form a complex (MRC) with C1QBP, which stabilizes MRE11/RAD50, while inhibiting MRE11 nuclease activity by preventing its binding to DNA or chromatin. Upon DNA damage, ATM phosphorylates MRE11-S676/S678 to quickly dissociate the MRC complex. Either excess or insufficient C1QBP impedes the recruitment of MRE11 to DSBs and impairs the DNA damage response. C1QBP is highly expressed in breast cancer and positively correlates with MRE11 expression, and the inhibition of C1QBP enhances tumor regression with chemotherapy. By influencing MRE11 at multiple levels, C1QBP is, thus, an important player in the DNA damage response.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Proteína Homóloga a MRE11/metabolismo , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido/genética , Animais , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Proteína Homóloga a MRE11/genética , Proteínas Mitocondriais/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Estabilidade Proteica , Células Sf9 , SpodopteraRESUMO
The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.
Assuntos
Aminoácidos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Recombinases/química , Recombinases/metabolismo , Recombinação Genética/genética , Aminoácidos/genética , Animais , Pareamento Incorreto de Bases , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Mutação , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP.
Assuntos
RNA Helicases DEAD-box , Replicação do DNA , Proteína do X Frágil da Deficiência Intelectual , Proteínas de Neoplasias , Estresse Fisiológico , Humanos , Cromatina/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA/biossíntese , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Hibridização de Ácido Nucleico , Estruturas R-Loop , RNA/química , RNA/metabolismoRESUMO
In this issue of Molecular Cell, Mazina et al. (2017) describe how the Rad52 protein mediates RNA-dependent DNA double-strand break repair via inverse strand exchange. This finding sheds light on how eukaryotes utilize RNA to repair chromosome breaks.
Assuntos
Reparo do DNA , Rad51 Recombinase/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Environmental agents like ionizing radiation (IR) and chemotherapeutic drugs can cause severe damage to the DNA, often in the form of double-strand breaks (DSBs). Remaining unrepaired, DSBs can lead to chromosomal rearrangements, and cell death. One major error-free pathway to repair DSBs is homologous recombination repair (HRR). Tousled-like kinase 1 (TLK1), a Ser/Thr kinase that regulates the DNA damage checkpoint, has been found to interact with RAD54, a central DNA translocase in HRR. To determine how TLK1 regulates RAD54, we inhibited or depleted TLK1 and tested how this impacts HRR in human cells using a ISce-I-GR-DsRed fused reporter endonuclease. Our results show that TLK1 phosphorylates RAD54 at three threonines (T41, T59 and T700), two of which are located within its N-terminal domain (NTD) and one is located within its C-terminal domain (CTD). Phosphorylation at both T41 and T59 supports HRR and protects cells from DNA DSB damage. In contrast, phosphorylation of T700 leads to impaired HRR and engenders no protection to cells from cytotoxicity and rather results in repair delay. Further, our work enlightens the effect of RAD54-T700 (RAD54-CTD) phosphorylation by TLK1 in mammalian system and reveals a new site of interaction with RAD51.