RESUMEN
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.
Asunto(s)
Proteína BRCA2 , Recombinasa Rad51 , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN de Cadena Simple/genética , ADN/metabolismo , Reparación del ADN , Unión ProteicaRESUMEN
Microhomology-mediated end joining (MMEJ) is an intrinsically mutagenic pathway of DNA double-strand break (DSB) repair essential for proliferation of homologous recombination (HR)-deficient tumors. Although targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as an MMEJ effector. We show that loss of APE2 inhibits MMEJ at deprotected telomeres and at intra-chromosomal DSBs and is epistatic with Pol Theta for MMEJ activity. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease activity, and further identify an uncharacterized domain required for its recruitment to DSBs. We conclude that this previously unappreciated role of APE2 in MMEJ contributes to the addiction of HRD cells to APE2, which could be exploited in the treatment of cancer.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Recombinación HomólogaRESUMEN
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.
Asunto(s)
ADN de Cadena Simple , Recombinasa Rad51 , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , ADN/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN , ADN de Cadena Simple/genética , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismoRESUMEN
POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.
Asunto(s)
Roturas del ADN de Doble Cadena , Neoplasias , Humanos , Replicación del ADN/genética , Inestabilidad Genómica , ADN de Cadena Simple/genética , Mutaciones Letales Sintéticas , Reparación del ADN por Unión de Extremidades , Neoplasias/genéticaRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Unión al ADN , Complejos Multiproteicos , Recombinasa Rad51 , Proteínas Supresoras de Tumor , Humanos , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Recombinación Homóloga , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Recombinasa Rad51/ultraestructura , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/ultraestructura , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Neoplasias/genética , Neoplasias/prevención & control , Proteómica , Simulación por Computador , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Roturas del ADN de Doble CadenaRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas Portadoras/genética , ADN de Helmintos/genética , Proteínas de Unión al ADN/genética , Recombinasa Rad51/genética , Reparación del ADN por Recombinación , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , ADN de Helmintos/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Unión Proteica , Recombinasa Rad51/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Transducción de Señal , Imagen Individual de MoléculaRESUMEN
DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3' to 5' polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2-4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Helicasas , Reparación del ADN , Recombinasa Rad51 , Proteína de Replicación A , ADN , ADN Helicasas/metabolismo , ADN de Cadena Simple , Recombinasa Rad51/metabolismo , Proteína de Replicación A/metabolismoRESUMEN
During prophase of the first meiotic division, cells deliberately break their DNA1. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes2. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism3-7. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over8, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points9. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes4.
Asunto(s)
Intercambio Genético , Endonucleasas/metabolismo , Meiosis , Homólogo 1 de la Proteína MutL/metabolismo , Proteínas MutL/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Cromosomas Humanos/genética , Secuencia Conservada , ADN/metabolismo , División del ADN , Enzimas Reparadoras del ADN/metabolismo , ADN Cruciforme/metabolismo , Exodesoxirribonucleasas/metabolismo , Humanos , Homólogo 1 de la Proteína MutL/química , Proteínas MutL/química , Proteínas MutS/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación C/metabolismoRESUMEN
To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2-deficient cells.
Asunto(s)
Proteína BRCA2/deficiencia , Roturas del ADN , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Línea Celular Tumoral , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Inestabilidad Genómica , Humanos , Proteína Homóloga de MRE11 , Factores de Transcripción/genéticaRESUMEN
To repair a DNA double-strand break (DSB) by homologous recombination (HR), the 5'-terminated strand of the DSB must be resected. The human MRE11-RAD50-NBS1 (MRN) and CtIP proteins were implicated in the initiation of DNA end resection, but the underlying mechanism remained undefined. Here, we show that CtIP is a co-factor of the MRE11 endonuclease activity within the MRN complex. This function is absolutely dependent on CtIP phosphorylation that includes the key cyclin-dependent kinase target motif at Thr-847. Unlike in yeast, where the Xrs2/NBS1 subunit is dispensable in vitro, NBS1 is absolutely required in the human system. The MRE11 endonuclease in conjunction with RAD50, NBS1, and phosphorylated CtIP preferentially cleaves 5'-terminated DNA strands near DSBs. Our results define the initial step of HR that is particularly relevant for the processing of DSBs bearing protein blocks.
Asunto(s)
Proteínas de Ciclo Celular/genética , Reparación del ADN por Unión de Extremidades/genética , ADN Helicasas/genética , Recombinación Homóloga/genética , Complejos Multiproteicos/genética , Ácido Anhídrido Hidrolasas , Proteínas Portadoras , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN , Endodesoxirribonucleasas , Humanos , Proteína Homóloga de MRE11 , Proteínas Nucleares , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
DNA end resection initiates DNA double-strand break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step via MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In the absence of NBS1, MRE11-RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP augments MRE11: a phosphorylation-dependent mode through NBS1 and a phosphorylation-independent mode without NBS1. In support, we show that limited DNA end resection occurs in vivo in the absence of the FHA and BRCT domains of NBS1. Collectively, our data suggest that NBS1 restricts the MRE11-RAD50 nuclease to S-G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/metabolismo , Ácido Anhídrido Hidrolasas , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas , Recombinación Homóloga , Humanos , Proteína Homóloga de MRE11/genética , Proteínas Nucleares/genética , FosforilaciónRESUMEN
DNA double-strand break repair by homologous recombination employs long-range resection of the 5' DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay between them has been shown, it remains unclear whether and how these proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single-molecule level and investigated Sgs1 regulation by Dna2, the single-stranded DNA-binding protein RPA, and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection both proteins form a functional complex and couple their activities. Sgs1 drives DNA unwinding and feeds single-stranded DNA to Dna2 for degradation. RPA was found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We hypothesize that the differential regulation of Sgs1 activity by its protein partners is important to support diverse cellular functions of Sgs1 during the maintenance of genome stability.
Asunto(s)
ADN/metabolismo , RecQ Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , ADN Helicasas/metabolismo , Reparación del ADN , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Inestabilidad Genómica , Saccharomyces cerevisiae/metabolismo , Imagen Individual de MoléculaRESUMEN
To repair a DNA double-strand break by homologous recombination, 5'-terminated DNA strands must first be resected to reveal 3'-overhangs. This process is initiated by a short-range resection catalyzed by MRE11-RAD50-NBS1 (MRN) stimulated by CtIP, which is followed by a long-range step involving EXO1 or DNA2 nuclease. DNA2 is a bifunctional enzyme that contains both single-stranded DNA (ssDNA)-specific nuclease and motor activities. Upon DNA unwinding by Bloom (BLM) or Werner (WRN) helicase, RPA directs the DNA2 nuclease to degrade the 5'-strand. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single-molecule biochemistry, we show that CtIP also dramatically stimulates the adenosine 5'-triphosphate (ATP) hydrolysis-driven motor activity of DNA2 involved in the long-range resection step. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. Accordingly, the stimulatory effect of CtIP is only observed with wild-type DNA2, but not the helicase-deficient variant. Similarly to the function of CtIP to promote MRN, also the DNA2 stimulatory effect is facilitated by CtIP phosphorylation. The domain of CtIP required to promote DNA2 is located in the central region lacking in lower eukaryotes and is fully separable from domains involved in the stimulation of MRN. These results establish how CtIP couples both MRE11-dependent short-range and DNA2-dependent long-range resection and define the involvement of the motor activity of DNA2 in this process. Our data might help explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.
Asunto(s)
ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/metabolismo , Reparación del ADN por Recombinación , Ácido Anhídrido Hidrolasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Pruebas de Enzimas , Hidrólisis , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteínas Recombinantes/metabolismo , Células Sf9RESUMEN
DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11-RAD50-NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM-DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350-600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN-CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550-600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.
Asunto(s)
Reparación del ADN , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/fisiología , Proteína BRCA1/metabolismo , Camptotecina/toxicidad , Línea Celular , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Endodesoxirribonucleasas/genética , Humanos , Dominios Proteicos , Eliminación de Secuencia , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
MutLγ, a heterodimer of the MutL homologues Mlh1 and Mlh3, plays a critical role during meiotic homologous recombination. The meiotic function of Mlh3 is fully dependent on the integrity of a putative nuclease motif DQHAX2EX4E, inferring that the anticipated nuclease activity of Mlh1-Mlh3 is involved in the processing of joint molecules to generate crossover recombination products. Although a vast body of genetic and cell biological data regarding Mlh1-Mlh3 is available, mechanistic insights into its function have been lacking due to the unavailability of the recombinant protein complex. Here we expressed the yeast Mlh1-Mlh3 heterodimer and purified it into near homogeneity. We show that recombinant MutLγ is a nuclease that nicks double-stranded DNA. We demonstrate that MutLγ binds DNA with a high affinity and shows a marked preference for Holliday junctions. We also expressed the human MLH1-MLH3 complex and show that preferential binding to Holliday junctions is a conserved capacity of eukaryotic MutLγ complexes. Specific DNA recognition has never been observed with any other eukaryotic MutL homologue. MutLγ thus represents a new paradigm for the function of the eukaryotic MutL protein family. We provide insights into the mode of Holliday junction recognition and show that Mlh1-Mlh3 prefers to bind the open unstacked Holliday junction form. This further supports the model where MutLγ is part of a complex acting on joint molecules to generate crossovers in meiosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ADN Cruciforme/metabolismo , ADN de Hongos/metabolismo , Desoxirribonucleasa I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Adaptadoras Transductoras de Señales/genética , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , ADN Cruciforme/genética , ADN de Hongos/genética , Desoxirribonucleasa I/genética , Humanos , Homólogo 1 de la Proteína MutL , Proteínas MutL , Multimerización de Proteína/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Homologous recombination (HR) plays a critical role in largely error-free repair of mitotic and meiotic DNA double-strand breaks (DSBs). DSBs are one of the most deleterious DNA lesions, which are repaired by non-homologous end joining (NHEJ), homologous recombination (HR) or, if compromised, micro-homology mediated end joining (MMEJ). If left unrepaired, DSBs can lead to cell death or if repaired incorrectly can result in chromosome rearrangements that drive cancer development. Here, we describe recent advances in the field of mitotic HR made using Caenorhabditis elegans roundworm, as a model system.
Asunto(s)
Caenorhabditis elegans , Reparación del ADN por Unión de Extremidades , Animales , Caenorhabditis elegans/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN , Recombinación Homóloga/genéticaRESUMEN
Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021).
Asunto(s)
ADN de Cadena Simple/química , ADN/química , Imagen Individual de Molécula/métodos , Proteínas Fluorescentes Verdes/química , Humanos , Imagen Óptica , Pinzas Ópticas , Recombinasa Rad51/química , Proteínas Recombinantes/química , Proteína de Replicación A/químicaRESUMEN
The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/metabolismo , Desnaturalización de Ácido Nucleico , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/biosíntesis , Transcripción Genética , Ácido Anhídrido Hidrolasas/genética , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Proteína Homóloga de MRE11/genética , Mutación , Proteínas Nucleares/genética , ARN Polimerasa II/genética , ARN Largo no Codificante/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.