RESUMO
Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.
Assuntos
Reparo do DNA/genética , Conversão Gênica , Rad51 Recombinase/metabolismo , Alelos , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos de Mamíferos/genética , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos , Feminino , Loci Gênicos , Recombinação Homóloga/genética , Homozigoto , Humanos , Mutação INDEL/genética , Camundongos Endogâmicos C57BL , Mosaicismo , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Ribonucleoproteínas/metabolismo , Zigoto/metabolismoRESUMO
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
Meiotic double-strand breaks (DSBs) are generated and repaired in a highly regulated manner to ensure formation of crossovers (COs) while also enabling efficient non-CO repair to restore genome integrity. We use structured-illumination microscopy to investigate the dynamic architecture of DSB repair complexes at meiotic recombination sites in relationship to the synaptonemal complex (SC). DSBs resected at both ends are converted into inter-homolog repair intermediates harboring two populations of BLM helicase and RPA, flanking a single population of MutSγ. These intermediates accumulate until late pachytene, when repair proteins disappear from non-CO sites and CO-designated sites become enveloped by SC-central region proteins, acquire a second MutSγ population, and lose RPA. These and other data suggest that the SC may protect CO intermediates from being dismantled inappropriately and promote CO maturation by generating a transient CO-specific repair compartment, thereby enabling differential timing and outcome of repair at CO and non-CO sites.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Reparo do DNA , Meiose , Recombinação Genética/genética , Complexo Sinaptonêmico/metabolismo , Animais , Caenorhabditis elegans/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Imageamento Tridimensional , Microscopia , Prófase , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Complexo Sinaptonêmico/químicaRESUMO
Tumor suppressor BRCA2 functions in homology-directed repair (HDR), the protection of stalled replication forks, and the suppression of replicative gaps, but their relative contributions to genome integrity and chemotherapy response are under scrutiny. Here, we report that mouse and human cells require a RAD51 filament stabilization motif in BRCA2 for fork protection and gap suppression but not HDR. In mice, the loss of fork protection/gap suppression does not compromise genome stability or shorten tumor latency. By contrast, HDR deficiency increases spontaneous and replication stress-induced chromosome aberrations and tumor predisposition. Unlike with HDR, fork protection/gap suppression defects are also observed in Brca2 heterozygous cells, likely due to reduced RAD51 stabilization at stalled forks/gaps. Gaps arise from PRIMPOL activity, which is associated with 5-hydroxymethyl-2'-deoxyuridine sensitivity due to the formation of SMUG1-generated abasic sites and is exacerbated by poly(ADP-ribose) polymerase (PARP) inhibition. However, HDR proficiency has the major role in mitigating sensitivity to chemotherapeutics, including PARP inhibitors.
Assuntos
Proteína BRCA2 , Replicação do DNA , Rad51 Recombinase , Animais , Humanos , Camundongos , Proteína BRCA2/metabolismo , Reparo do DNA , Instabilidade Genômica , Genômica , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por RecombinaçãoRESUMO
Centromeres are essential for chromosome segregation in most animals and plants yet are among the most rapidly evolving genome elements. The mechanisms underlying this paradoxical phenomenon remain enigmatic. Here, we report that human centromeres innately harbor a striking enrichment of DNA breaks within functionally active centromere regions. Establishing a single-cell imaging strategy that enables comparative assessment of DNA breaks at repetitive regions, we show that centromeric DNA breaks are induced not only during active cellular proliferation but also de novo during quiescence. Markedly, centromere DNA breaks in quiescent cells are resolved enzymatically by the evolutionarily conserved RAD51 recombinase, which in turn safeguards the specification of functional centromeres. This study highlights the innate fragility of centromeres, which may have been co-opted over time to reinforce centromere specification while driving rapid evolution. The findings also provide insights into how fragile centromeres are likely to contribute to human disease.
Assuntos
Centrômero , DNA , Animais , Humanos , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinação GenéticaRESUMO
Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.
Assuntos
Proteína BRCA2 , Rad51 Recombinase , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA de Cadeia Simples/genética , DNA/metabolismo , Reparo do DNA , Ligação ProteicaRESUMO
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
Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Rad51 Recombinase/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
DNA recombinases face the daunting task of locating and pairing up specific sequences among millions of base pairs in a genome, all within about an hour. Qi et al. show that recombinases solve this problem by searching in 8-nt microhomology units, reducing the search space and accelerating the homology search.
Assuntos
Recombinação Homóloga , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs)1,2. However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.
Assuntos
Microscopia Crioeletrônica , Quebras de DNA de Cadeia Dupla , Nucleossomos , Rad51 Recombinase , Proteínas de Saccharomyces cerevisiae , Humanos , DNA/química , DNA/metabolismo , DNA/ultraestrutura , Reparo do DNA/genética , Nucleossomos/química , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Rad51 Recombinase/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Mutação , Domínios Proteicos , Histonas/química , Histonas/metabolismo , Histonas/ultraestrutura , Ligação ProteicaRESUMO
The efficiency of homologous recombination (HR) in the repair of DNA double-strand breaks (DSBs) is closely associated with genome stability and tumor response to chemotherapy. While many factors have been functionally characterized in HR, such as TOPBP1, their precise regulation remains unclear. Here, we report that TOPBP1 interacts with the RNA-binding protein HTATSF1 in a cell-cycle- and phosphorylation-dependent manner. Mechanistically, CK2 phosphorylates HTATSF1 to facilitate binding to TOPBP1, which promotes S-phase-specific TOPBP1 recruitment to damaged chromatin and subsequent RPA/RAD51-dependent HR, genome integrity, and cancer-cell viability. The localization of HTATSF1-TOPBP1 to DSBs is potentially independent of the transcription-coupled RNA-binding and processing capacity of HTATSF1 but rather relies on the recognition of poly(ADP-ribosyl)ated RPA by HTATSF1, which can be blunted with PARP inhibitors. Together, our study provides a mechanistic insight into TOPBP1 loading at HR-prone DSB sites via HTATSF1 and reveals how RPA-RAD51 exchange is tuned by a PARylation-phosphorylation cascade.
Assuntos
Poli ADP Ribosilação , Rad51 Recombinase , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga/genética , Fosforilação , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismoRESUMO
Oncogene activation during tumorigenesis promotes DNA replication stress (RS), which subsequently drives the formation of cancer-associated chromosomal rearrangements. Many episodes of physiological RS likely arise due to conflicts between the DNA replication and transcription machineries operating simultaneously at the same loci. One role of the RAD51 recombinase in human cells is to protect replication forks undergoing RS. Here, we have identified a key role for RAD51 in preventing transcription-replication conflicts (TRCs) from triggering replication fork breakage. The genomic regions most affected by RAD51 deficiency are characterized by being replicated and transcribed in early S-phase and show significant overlap with loci prone to cancer-associated amplification. Consistent with a role for RAD51 in protecting against transcription-replication conflicts, many of the adverse effects of RAD51 depletion are ameliorated by inhibiting early S-phase transcription. We propose a model whereby RAD51 suppresses fork breakage and subsequent inadvertent amplification of genomic loci prone to experiencing TRCs.
Assuntos
Replicação do DNA , Rad51 Recombinase , Cromossomos/metabolismo , Humanos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Fase S/genética , Transcrição GênicaRESUMO
RAD51 and the breast cancer suppressor BRCA2 have critical functions in DNA double-strand (dsDNA) break repair by homologous recombination and the protection of newly replicated DNA from nucleolytic degradation. The recombination function of RAD51 requires its binding to single-stranded DNA (ssDNA), whereas binding to dsDNA is inhibitory. Using reconstituted MRE11-, EXO1-, and DNA2-dependent nuclease reactions, we show that the protective function of RAD51 unexpectedly depends on its binding to dsDNA. The BRC4 repeat of BRCA2 abrogates RAD51 binding to dsDNA and accordingly impairs the function of RAD51 in protection. The BRCA2 C-terminal RAD51-binding segment (TR2) acts in a dominant manner to overcome the effect of BRC4. Mechanistically, TR2 stabilizes RAD51 binding to dsDNA, even in the presence of BRC4, promoting DNA protection. Our data suggest that RAD51's dsDNA-binding capacity may have evolved together with its function in replication fork protection and provide a mechanistic basis for the DNA-protection function of BRCA2.
Assuntos
DNA de Cadeia Simples , Rad51 Recombinase , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismoRESUMO
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
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 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
In this issue of Molecular Cell, Roy et al. (2021) and Belan et al. (2021) demonstrate that the yeast and nematode RAD51 paralog complexes function as chaperones to promote the assembly of the RAD51 nucleoprotein filament on RPA-coated ssDNA.
Assuntos
Rad51 Recombinase , Proteínas de Saccharomyces cerevisiae , DNA de Cadeia Simples/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de MoléculaRESUMO
Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a "chaperone" to promote 3' to 5' filament growth via highly dynamic engagement with 5' filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Transporte/genética , DNA de Helmintos/genética , Proteínas de Ligação a DNA/genética , Rad51 Recombinase/genética , Reparo de DNA por Recombinação , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Helmintos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Rad51 Recombinase/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Transdução de Sinais , Imagem Individual de MoléculaRESUMO
The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication.
Assuntos
Proteína BRCA2/genética , Replicação do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a RNA/genética , Rad51 Recombinase/genética , Trifosfato de Adenosina/metabolismo , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Hidrólise , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Ligação a RNA/metabolismo , Rad51 Recombinase/metabolismo , Transdução de Sinais , Imagem Individual de Molécula , Proteína Vermelha FluorescenteRESUMO
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.