RESUMEN
The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3'-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.
Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Roturas del ADN de Doble Cadena , ADN , Humanos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , ADN/metabolismo , ADN/genética , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Edición Génica , Endonucleasas/metabolismo , Endonucleasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Reparación del ADN por Unión de Extremidades , División del ADN , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.
Asunto(s)
División del ADN , Paramecium , Paramecium/genética , Paramecium/metabolismo , Roturas del ADN de Doble Cadena , Genoma de Protozoos , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Reparación del ADN , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Reparación del ADN por Unión de ExtremidadesRESUMEN
The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.
Asunto(s)
Proteínas de Unión al ADN , Ácido Fítico , Animales , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/genética , Autoantígeno Ku/metabolismo , Mamíferos/genética , HumanosRESUMEN
The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ultimately lead to diseases including cancer. Non-homologous end joining (NHEJ) is one of two core mechanisms utilized to repair DSBs. DNA-PK is a key component in this process and has recently been shown to form alternate long-range synaptic dimers. This has led to the proposal that these complexes can be formed before transitioning to a short-range synaptic complex. Here we present cryo-EM data representing an NHEJ supercomplex consisting of a trimer of DNA-PK in complex with XLF, XRCC4, and DNA Ligase IV. This trimer represents a complex of both long-range synaptic dimers. We discuss the potential role of the trimeric structure, and possible higher order oligomers, as structural intermediates in the NHEJ mechanism, or as functional DNA repair centers.
Asunto(s)
Enzimas Reparadoras del ADN , Reparación del ADN , Humanos , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Microscopía por Crioelectrón , Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP) , Proteína Quinasa Activada por ADN/metabolismo , ADN/genéticaRESUMEN
Nonhomologous end joining is a critical mechanism that repairs DNA double-strand breaks in human cells. In this work, we address the structural and functional role of the accessory protein PAXX [paralog of x-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor (XLF)] in this mechanism. Here, we report high-resolution cryo-electron microscopy (cryo-EM) and x-ray crystallography structures of the PAXX C-terminal Ku-binding motif bound to Ku70/80 and cryo-EM structures of PAXX bound to two alternate DNA-dependent protein kinase (DNA-PK) end-bridging dimers, mediated by either Ku80 or XLF. We identify residues critical for the Ku70/PAXX interaction in vitro and in cells. We demonstrate that PAXX and XLF can bind simultaneously to the Ku heterodimer and act as structural bridges in alternate forms of DNA-PK dimers. Last, we show that engagement of both proteins provides a complementary advantage for DNA end synapsis and end joining in cells.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN , Humanos , Microscopía por Crioelectrón , ADN , Enzimas Reparadoras del ADN/genéticaRESUMEN
Nonhomologous end-joining (NHEJ) factors act in replication-fork protection, restart, and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish the NHEJ factor Ku-mediated barrier to nascent strand degradation in fission yeast. RNase H activities promote nascent strand degradation and replication restart, with a prominent role of RNase H2 in processing RNA:DNA hybrids to overcome the Ku barrier to nascent strand degradation. RNase H2 cooperates with the MRN-Ctp1 axis to sustain cell resistance to replication stress in a Ku-dependent manner. Mechanistically, the need of RNaseH2 in nascent strand degradation requires the primase activity that allows establishing the Ku barrier to Exo1, whereas impairing Okazaki fragment maturation reinforces the Ku barrier. Finally, replication stress induces Ku foci in a primase-dependent manner and favors Ku binding to RNA:DNA hybrids. We propose a function for the RNA:DNA hybrid originating from Okazaki fragments in controlling the Ku barrier specifying nuclease requirement to engage fork resection.
Asunto(s)
ARN , Schizosaccharomyces , ARN/genética , ARN/metabolismo , ADN Primasa/metabolismo , ADN/genética , ADN/metabolismo , Replicación del ADN , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ribonucleasas/genéticaAsunto(s)
Adenosina Trifosfato/metabolismo , Reparación de la Incompatibilidad de ADN , ADN/metabolismo , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Daño del ADN , Hidrólisis , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/química , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/ultraestructura , Conformación ProteicaRESUMEN
We developed an Xrcc4M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4-deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core non-homologous end-joining (NHEJ) DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4M61R mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double Knock Out (DKO) settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights into the understanding of the clinical manifestations of human XRCC4-deficient condition, in particular its absence of immune deficiency.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/genética , Linfocitos/inmunología , Mutación Missense , Inmunodeficiencia Combinada Grave/genética , Recombinación V(D)J , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/metabolismoRESUMEN
Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.
Asunto(s)
ADN Ligasa (ATP)/ultraestructura , Enzimas Reparadoras del ADN/ultraestructura , Proteína Quinasa Activada por ADN/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Complejos Multiproteicos/ultraestructura , Apoptosis/genética , Microscopía por Crioelectrón , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/genética , ADN Ligasa (ATP)/genética , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/ultraestructura , Complejos Multiproteicos/genética , Fosforilación/genéticaRESUMEN
FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding. Disruption of the FAN1-MLH1 interaction confers cellular hypersensitivity to ICL damage and defective repair of CAG/CTG slip-outs, intermediates of repeat expansion mutations. FAN1-S126 phosphorylation, which hinders FAN1-MLH1 association, is cell cycle-regulated by cyclin-dependent kinase activity and attenuated upon ICL induction. Our data highlight the FAN1-MLH1 complex as a phosphorylation-regulated determinant of ICL response and repeat stability, opening novel paths to modify cancer and neurodegeneration.
Asunto(s)
Endodesoxirribonucleasas , Exodesoxirribonucleasas , ADN , Daño del ADN , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Enzimas Multifuncionales/genéticaRESUMEN
In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ's dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.
Asunto(s)
Reparación de la Incompatibilidad de ADN/fisiología , Endonucleasas/metabolismo , Homólogo 1 de la Proteína MutL/metabolismo , Proteínas MutL/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endonucleasas/química , Meiosis , Modelos Moleculares , Homólogo 1 de la Proteína MutL/química , Homólogo 1 de la Proteína MutL/genética , Proteínas MutL/química , Proteínas MutL/genética , Reparación del ADN por Recombinación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencias de Aminoácidos , Cromosomas Fúngicos/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/química , Exodesoxirribonucleasas/química , Modelos Moleculares , Complejos Multiproteicos , Mutación , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genéticaRESUMEN
DNA double-strand breaks (DSBs) are accidental lesions generated by various endogenous or exogenous stresses. DSBs are also genetically programmed events during the V(D)J recombination process, meiosis, or other genome rearrangements, and they are intentionally generated to kill cancer during chemo- and radiotherapy. Most DSBs are processed in mammalian cells by the classical nonhomologous end-joining (c-NHEJ) pathway. Understanding the molecular basis of c-NHEJ has major outcomes in several fields, including radiobiology, cancer therapy, immune disease, and genome editing. The heterodimer Ku70/80 (Ku) is a central actor of the c-NHEJ as it rapidly recognizes broken DNA ends in the cell and protects them from nuclease activity. It subsequently recruits many c-NHEJ effectors, including nucleases, polymerases, and the DNA ligase 4 complex. Beyond its DNA repair function, Ku is also involved in several other DNA metabolism processes. Here, we review the structural and functional data on the DNA and RNA recognition properties of Ku implicated in DNA repair and in telomeres maintenance.
Asunto(s)
Autoantígeno Ku/metabolismo , Animales , Reparación del ADN , Evolución Molecular , Humanos , Autoantígeno Ku/química , Autoantígeno Ku/genética , Procesamiento Proteico-PostraduccionalRESUMEN
Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( https://mosbio.sciencesconf.org/ ). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.
Asunto(s)
Sustancias Macromoleculares/análisis , Calorimetría , ADN , Humanos , Ligandos , ProteínasRESUMEN
We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a â¼2 s synapsis between blunt DNA ends that is increased to â¼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Reparación del ADN por Unión de Extremidades , Autoantígeno Ku/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/química , ADN/metabolismo , ADN Ligasas/metabolismo , Autoantígeno Ku/química , Multimerización de ProteínaRESUMEN
Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation.
Asunto(s)
ADN , Imagen Individual de Molécula , Microscopía por Crioelectrón , Sustancias Macromoleculares , Microscopía ElectrónicaRESUMEN
Interactions between protein complexes and DNA are central regulators of the cell life. They control the activation and inactivation of a large set of nuclear processes including transcription, replication, recombination, repair, and chromosome structures. In the literature, protein-DNA interactions are characterized by highly complementary approaches including large-scale studies and analyses in cells. Biophysical approaches with purified materials help to evaluate if these interactions are direct or not. They provide quantitative information on the strength and specificity of the interactions between proteins or protein complexes and their DNA substrates. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) are widely used and are complementary methods to characterize nucleo-protein complexes and quantitatively measure protein-DNA interactions. We present here protocols to analyze the interactions between a DNA repair complex, Ku70-Ku80 (Ku) (154 kDa), and DNA substrates. ITC is a label-free method performed with both partners in solution. It serves to determine the dissociation constant (Kd), the enthalpy (ΔH), and the stoichiometry N of an interaction. MST is used to measure the Kd between the protein or the DNA labeled with a fluorescent probe. We report the data obtained on Ku-DNA interactions with ITC and MST and discuss advantages and drawbacks of both the methods.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Sustancias Macromoleculares/química , Fenómenos Bioquímicos , Calorimetría , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Conformación Molecular , Unión Proteica , Relación Estructura-Actividad , TermodinámicaRESUMEN
Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1-Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ-Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.