Добірка наукової літератури з теми "Filaments Rad51"

Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as a template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52–Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, in vivo and in vitro analyzes of these mutants indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

The bypass of DNA lesions that block replicative polymerases during DNA replication relies on DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway depends on specialized DNA polymerases that incorporate nucleotides in front of base lesions, potentially inducing mutagenesis. Two error-free pathways can bypass the lesions: the template switching pathway, which uses the sister chromatid as a template, and the homologous recombination pathway (HR), which also can use the homologous chromosome as template. The balance between error-prone and error-free pathways controls the mutagenesis level. Therefore, it is crucial to precisely characterize factors that influence the pathway choice to better understand genetic stability at replication forks. In yeast, the complex formed by the Rad51 paralogs Rad55 and Rad57 promotes HR and template-switching at stalled replication forks. At DNA double-strand breaks (DSBs), this complex promotes Rad51 filament formation and stability, notably by counteracting the Srs2 anti-recombinase. To explore the role of the Rad55-Rad57 complex in error-free pathways, we monitored the genetic interactions between Rad55-Rad57, the translesion polymerases Polζ or Polη, and Srs2 following UV radiation that induces mostly single-strand DNA gaps. We found that the Rad55-Rad57 complex was involved in three ways. First, it protects Rad51 filaments from Srs2, as it does at DSBs. Second, it promotes Rad51 filament stability independently of Srs2. Finally, we observed that UV-induced HR is almost abolished in Rad55-Rad57 deficient cells, and is partially restored upon Polζ or Polη depletion. Hence, we propose that the Rad55-Rad57 complex is essential to promote Rad51 filament stability on single-strand DNA gaps, notably to counteract the error-prone TLS polymerases and mutagenesis.

Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 “anti-recombinase” restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we cytologically characterize Srs2 function in vivo and describe a novel mechanism for regulating the initiation of HR. We find that Srs2 is recruited separately to replication and repair centers and identify the genetic requirements for recruitment. In the absence of Srs2 activity, Rad51 foci accumulate, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2 removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR.

Homologous recombination (HR) is a DNA repair mechanism of double-strand breaks and blocked replication forks, involving a process of homology search leading to the formation of synaptic intermediates that are regulated to ensure genome integrity. RAD51 recombinase plays a central role in this mechanism, supported by its RAD52 and BRCA2 partners. If the mediator function of BRCA2 to load RAD51 on RPA-ssDNA is well established, the role of RAD52 in HR is still far from understood. We used transmission electron microscopy combined with biochemistry to characterize the sequential participation of RPA, RAD52, and BRCA2 in the assembly of the RAD51 filament and its activity. Although our results confirm that RAD52 lacks a mediator activity, RAD52 can tightly bind to RPA-coated ssDNA, inhibit the mediator activity of BRCA2, and form shorter RAD51-RAD52 mixed filaments that are more efficient in the formation of synaptic complexes and D-loops, resulting in more frequent multi-invasions as well. We confirm the in situ interaction between RAD51 and RAD52 after double-strand break induction in vivo. This study provides new molecular insights into the formation and regulation of presynaptic and synaptic intermediates by BRCA2 and RAD52 during human HR.

The proper maintenance of genetic material is essential for the survival of living organisms. One of the main safeguards of genome stability is homologous recombination involved in the faithful repair of DNA double-strand breaks, the restoration of collapsed replication forks, and the bypass of replication barriers. Homologous recombination relies on the formation of Rad51 nucleoprotein filaments which are responsible for the homology-based interactions between DNA strands. Here, we demonstrate that without the regulation of these filaments by Srs2 and Rad54, which are known to remove Rad51 from single-stranded and double-stranded DNA, respectively, the filaments strongly inhibit damage-associated DNA synthesis during DNA repair. Furthermore, this regulation is essential for cell survival under normal growth conditions, as in the srs2Δ rad54Δ mutants, unregulated Rad51 nucleoprotein filaments cause activation of the DNA damage checkpoint, formation of mitotic bridges, and loss of genetic material. These genome instability features may stem from the problems at stalled replication forks as the lack of Srs2 and Rad54 in the presence of Rad51 nucleoprotein filaments impedes cell recovery from replication stress. This study demonstrates that the timely and efficient disassembly of recombination machinery is essential for genome maintenance and cell survival.

ABSTRACT The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B −/− cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B −/− cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to γ-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B −/−cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.

Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5–Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.

In eukaryotes, Rad51 and Rad54 functionally cooperate to mediate homologous recombination and the repair of damaged chromosomes by recombination. Rad51, the eukaryotic counterpart of the bacterial RecA recombinase, forms filaments on single-stranded DNA that are capable of pairing the bound DNA with a homologous double-stranded donor to yield joint molecules. Rad54 enhances the homologous DNA pairing reaction, and this stimulatory effect involves a physical interaction with Rad51. Correspondingly, the ability of Rad54 to hydrolyze ATP and introduce superhelical tension into covalently closed circular plasmid DNA is stimulated by Rad51. By controlled proteolysis, we show that the amino-terminal region of yeast Rad54 is rather unstructured. Truncation mutations that delete the N-terminal 113 or 129 amino acid residues of Rad54 attenuate or ablate physical and functional interactions with Rad51 under physiological ionic strength, respectively. Surprisingly, under less stringent conditions, the Rad54 Δ129 protein can interact with Rad51 in affinity pull-down and functional assays. These results highlight the functional importance of the N-terminal Rad51 interaction domain of Rad54 and reveal that Rad54 contacts Rad51 through separable epitopes.

Abstract RADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using single-molecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

Abstract Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.

La recombinaison homologue (RH) est une des voies majeures de réparation des dommages de l'ADN telles que les cassures double brin (CDB). Cette voie est également impliquée dans le redémarrage des fourches de réplication bloquées à une lésion. Une étape clé de cette voie de réparation consiste en la formation de filaments nucléoprotéiques formés de la recombinase Rad51 sur de l'ADN simple brin (ADNsb). Ces nucléofilaments sont responsables de la recherche d'homologie et de l'invasion d'un ADN intact et semblable à l'ADN endommagé afin de l'utiliser comme matrice pour la synthèse réparatrice. La formation de ces nucléofilaments est finement régulée. Chez la levure Saccharomyces cerevisiae, la formation des filaments Rad51 est assurée par les régulateurs positifs que sont la protéine médiatrice Rad52 et les deux complexes composés de protéines paralogues de Rad51, le complexe Rad55-Rad57 et le complexe SHU. Ils jouent également un rôle de protection de ce nucléofilament face au régulateur négatif Srs2 dont ils contrebalancent les effets de désassemblage. Pour mieux comprendre le mécanisme de régulation du nucléofilament Rad51, une connaissance plus approfondie des interactions complexes intervenant entre ces multiples acteurs est nécessaire. Durant ma thèse, nous avons cherché à déterminer la structure des complexes de paralogues de Rad51 en association avec les filaments de Rad51. Pour cela, nous avons combiné une approche bio-informatique structurale, basée sur des alignements de séquences et la structure publiée de Rad51, avec des expériences de double-hybride de levure (Y2H). Cette stratégie nous a permis de proposer pour la première fois un modèle pour l'organisation des complexes de paralogues de Rad51, les complexes Rad55-Rad57 et SHU, en association avec la recombinase Rad51. Ce modèle a été validé par des analyses génétiques de mutations perturbant chaque domaine d'interaction. Dans notre modèle, Rad55-Rad57 adopte une structure similaire à celle d'un dimère de Rad51 et n'interagit qu'avec l'extrémité 5' des filaments de Rad51 et uniquement par l'intermédiaire de la sous-unité Rad57. Nos analyses génétiques suggèrent que le rôle principal de l'interaction entre Rad55-Rad57 et Rad51 est de protéger les filaments Rad51 contre la translocase Srs2. De l'autre côté de Rad55-Rad57, Rad55 interagit avec la sous-unité Csm2 du complexe SHU par son extrémité N-terminale. Il est intéressant de noter que nos analyses génétiques ont révélé que SHU et l'extrémité N-terminale de Rad55 sont dispensables pour la réparation des CDB. Ils sont en revanche impliqués dans la réparation des brèches simple brin induites par les UV. Nous proposons que le complexe SHU stabilise la liaison de Rad55-Rad57 sur les brèches simple brin, favorisant ainsi une meilleure stabilité des filaments de Rad51. Ainsi, les filaments Rad51 seraient plus résistants à l'activité déstabilisatrice de l'hélicase anti-recombinase Srs2 et permettraient à la RH de rivaliser avec d'autres voies de tolérance aux dommages de l'ADN impliquant des ADN polymérases translésionnelles responsables de l'incorporation de mutations. Nos données permettent de proposer un modèle d'installation du filament Rad51 par les complexes de paralogues, Rad55-Rad57 et SHU, en collaboration avec Rad52
Homologous recombination (HR) is one of the major repair pathways for DNA damage such as double-strand breaks (DSBs). This pathway is also involved in restarting replication forks stalled by DNA lesion. A key step in this repair pathway involves the formation of nucleoprotein filaments formed by Rad51 recombinase on single-stranded DNA (ssDNA). These nucleofilaments are responsible for homology search and invasion of an intact DNA similar to the damaged DNA in order to use it as a template for repair synthesis. The formation of these nucleofilaments is tightly regulated. In the budding yeast Saccharomyces cerevisiae, the formation of Rad51 filaments is ensured by positive regulators such as the Rad52 mediator protein and the two complexes composed of Rad51 paralogue proteins, the Rad55-Rad57 complex and the SHU complex. They also play a role in protecting this nucleofilament from the negative regulator Srs2 by counterbalancing its disassembly effects. To gain a better understanding of the regulatory mechanism of the Rad51 nucleofilament, we need a more detailed understanding of the complex interactions between these multiple players. During my thesis, we aimed at determining the structure of Rad51 paralog complexes in association with Rad51 filaments. To do this, we combined a structural bioinformatics approach, based on sequence alignments and the published structure of Rad51, with yeast two-hybrid (Y2H) experiments. This strategy allowed us to build for the first time a model for the organisation of Rad51 paralog complexes, the Rad55-Rad57 and SHU complexes, in association with the Rad51 recombinase. This model was further validated by genetic analysis of mutations disrupting each interaction domain. In our model, Rad55-Rad57 adopts a similar structure than a dimer of Rad51 and it interacts only with the 5'-end of Rad51 filaments and only through the Rad57 subunit. Our genetic analyses suggest that the major role of the interaction between Rad55-Rad57 and Rad51 is to protect Rad51 filaments against the Srs2 translocase. On the other side of Rad55-Rad57, Rad55 interacts with the Csm2 subunit of the SHU complex through its N-terminal end. Interestingly, our genetic analyses revealed that SHU and the N-terminal end of Rad55 are dispensable for DSB repair. However, they are involved in the repair of UV-induced single-strand breaks. We propose that the SHU complex stabilizes the binding of Rad55-Rad57 on ssDNA gaps, thereby promoting enhanced stability of Rad51 filaments. Thus, Rad51 filaments would be more resistant to the destabilising activity of the Srs2 translocase and would allow HR to compete with alternative gap filling pathways involving error-prone translesion DNA polymerases. Our data allow us to propose a model for the installation of the Rad51 filament by paralog complexes, Rad55-Rad57 and SHU, in collaboration with Rad52

Dans cette thèse, nous avons étudié plusieurs aspects des interactions entre l’ADN et la protéine Rad51 humaine, qui est l’acteur clé du processus de recombinaison homologue eucaryote (échange de brins de séquences homologues entre deux molécules d’ADN). Cette protéine polymérise pour former des filaments nucléoprotéiques hélicoïdaux au sein desquels, dans leur conformation « canonique », l’ADN est surétiré et sous-enroulé. La technique de pinces magnétiques utilisée permet de manipuler un filament individuel en exerçant une force et une contrainte de surenroulement aux extrémités de la molécule d’ADN, tout en mesurant son extension bout à bout. Nous avons étudié l’influence de la concentration de la protéine en solution sur la cinétique de formation d’un filament ; nos résultats révèlent que l’élongation du filament procède par un mécanisme de nucléations multiples dont l’étape cinétiquement limitante est la formation d’un oligomère Rad51 impliquant 5 ou 6 monomères. Nous avons également mis au point un protocole permettant d’observer l’échange de brins au sein d’un filament piégé dans les pinces. Par ailleurs, nous nous sommes intéressés à la dynamique des filaments Rad51-ADN double brin en présence d’une contrainte de torsion. La modification de la réponse force-extension du filament induite par une torsion positive ne peut s’expliquer par un modèle d’élasticité de torsion tel que celui utilisé pour l’ADN double brin seul ; nous proposons un modèle dans lequel, sous un couple de torsion positif, le filament Rad51-ADN subit une transition réversible entre la forme « canonique » et une seconde conformation dans laquelle l’ADN n’est que faiblement étiré et sous-enroulé

La recombinaison homologue (RH) est essentielle à la réparation des cassures double-brin de l'ADN, mais elle peut être une source d'instabilité génétique et doit être strictement contrôlée. Il a été montré chez la levure que certains intermédiaires de RH sont létaux et que l’hélicase Srs2 a pour fonction de les éliminer. Cet aspect de la toxicité de la RH est mal connu. J’ai montré au cours de ma thèse que les structures toxiques de RH induites par des agents génotoxiques ou par des blocages de réplication dans les cellules haploïdes sont des filaments nucléoprotéiques Rad51 probablement non fonctionnels. Lors du processus normal de la RH, Rad51 permet à l’ADN lésé de trouver et d’envahir une séquence d’ADN homologue utilisée comme matrice pour la synthèse d’ADN réparatrice. J’ai également montré que certains mutants de Rad52, une protéine essentielle à la formation des filaments Rad51, éliminent leur toxicité potentielle. L’étude de certains de ces mutants a permis de montrer une modification des activités catalytiques de Rad52, notamment son activité médiatrice. L’induction massive de la sumoylation de Rad52 produit le même effet. D’autre part, un des mutants identifiés, rad52-P381S, a perdu l’interaction avec Rad51 sans affecter la RH. Dans le contexte de ce mutant, la RH est strictement dépendante de RAD55. Toutefois, dans les cellules rad52Δ, il n’y a pas de formation de filaments Rad51. Ceci montre que Rad55 peut former des filaments Rad51 en coordination avec Rad52, même si ce dernier ne peut pas agir lui même avec Rad51. L’étude de ces mutants va permettre de mieux comprendre le contrôle de la formation des filaments Rad51 par les médiateurs et les hélicases
Homologous recombination (HR) is essential for double-strand break repair and participates in post-replication restart of stalled and collapsed replication forks. However, HR can lead to genome rearrangements and has to be strictly controlled. The budding yeast Srs2 is involved in the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated several alleles of RAD52, a gene that codes for the most central recombination protein in yeast. Interestingly, we observed that these mutants bypass the requirement for Srs2 without affecting DNA repair by HR. The genetic study of one of these mutants (rad52-L264P) showed that it specifically prevents the formation of unproductive Rad51 filaments before strand invasion, allowing us to define Srs2 substrates. The avoidance of toxic Rad51 filaments can also be overcome by stimulating Rad52 sumoylation. Further analysis showed that some of the mutated Rad52 proteins and the sumoylated form of Rad52 are characterized by a modified mediator activity. One of these mutants, rad52-P381S have lost its interaction with Rad51 without affecting HR. In this mutant, HR is strictly dependent on RAD55. However, Rad51 filaments are not formed in rad52Δ cells. Altogether these results show that Rad55 can assemble Rad51 filaments in coordination with Rad52, even though Rad52 cannot interact with Rad51. The study of these mutants will help to better understand how the mediators and helicases regulate the Rad51 filaments formation and its characteristics

碩士
國立臺灣大學
化學研究所
105
Homologous recombination catalyzed by RAD51 recombinases is a crucial DNA repair pathway in eukaryotes. In the presence of ATP, RAD51 assembles on single-stranded DNA to form nucleoprotein filaments, and initiates homologous recombinational repair of DNA double-stranded breaks. The SWI5-SFR1 complex has been found to regulate RAD51 filament assembly and enhance strand exchange activity, but the detailed mechanism is not clear. Here we improved our home-built optical tweezers platform to 1 nm resolution, and utilized it to study the assembly and disassembly dynamics of mRAD51 filaments in the presence of SWI5-SFR1 complex. In the case of double-stranded DNA, mRAD51 assembly process is stimulated in the prescence of SWI5-SFR1, but the disassembly process is not affected. On the other hand, mRAD51 assembles onto single-stranded DNA with an enhanced rate in the prescence of SWI5-SFR1, and the disassembly process from ssDNA is suppressed by the SWI5-SFR1 complex. This indicates that the SWI5-SFR1 stabilization function happens in the mRAD51 nucleoprotein filament formation onto single-stranded DNA, while SWI5-SFR1 only alters the kext of double-stranded DNA filament formation, not kdis. These regulatory functions of SWI5-SFR1 imply not only efficient stabilization of mRAD51 nucleoprotein filament during strand exchange, but also offer efficient mRAD51 turnover once the reaction is completed.

碩士
國立臺灣大學
化學研究所
100
Rad51 recombinases in eukaryotes and RecA recombinases in prokaryotes play an essential role in repairing damaged DNA by the homologous recombinational repair pathway. Once assembled on single-stranded (ss) DNA, Rad51 nucleoprotein filaments mediate the pairing and strand exchange with the homologous sequence. Single-molecule tethered particle motion (TPM) experiments monitor the DNA length and topology change during biochemical processes, and allow us to study the mechanistic details of DNA recombination processes. In the Rad51 invading strand experiments, beads were labeled on the invading ssDNA with sequence homologous to the surface-anchored duplex DNA. When bead-labeled Rad51 nucleoprotein filaments first interacted with the surface-anchored DNA, the initial Brownian motion (BM) amplitude which was resulted from the combined length contribution of the Rad51 nucleoprotein filament and surface anchored duplex DNA can be detected. The initial Brownian motion provides information on the initial contact point of the Rad51 nucleoprotein filament as well as its polarity preference for the stable synapses formation. A sum of two-segment model successfully describes the distribution of initial BM values. For transient events, the synaptic complex formation initiated at random position with no end preference. For events that drive successfully into the final strand exchange product, 5′-end segment of Rad51 nucleoprotein filament was preferentially used. Our studies suggest that Rad51 nucleoprotein filaments carry out initial strand exchange in the 5′-to-3′ direction.

Homologous recombination (HR) is an essential mechanism for accurate repair of DNA double-strand breaks (DSBs). However, HR must be tightly controlled because excessive or unwanted HR events can lead to genome instability, which is a prerequisite for premature aging and cancer development. A critical step of HR is the loading of RAD51 molecules onto single-stranded DNA regions generated in the vicinity of the DSB, leading to the formation of a nucleoprotein filament. Several DNA helicases have been involved in the regulation of the HR process. One of these is human FBH1 (F-box DNA helicase 1) that is a member of SF1 superfamily of helicases. As a unique DNA helicase, FBH1 additionally possesses a conserved F-box motif that allows it to assemble into an SCF complex, an E3 ubiquitin ligase that targets proteins for degradation. FBH1 has been implicated in the restriction of nucleoprotein filament stability. However, the exact mechanism of how FBH1 controls the RAD51 action is still not certain. In this work, we revealed that FBH1 actively disassembles RAD51 nucleoprotein filament. We also show that FBH1 interacts with RAD51 and RPA physically in vitro. Based on these data, we propose a potential mechanism of FBH1 antirecombinase function.

碩士
國立臺灣大學
化學研究所
107
Dmc1 and Rad51 recombinases play important roles in the DNA double strand break repair. During the homologous recombination, recombinase binds to the resected damaged DNA to form a nucleoprotein filament, responsible for homology pairing and strand exchange. Rad51 and Dmc1 both exist in most eukaryotic cells, sharing similar amino acid sequences, structures and functions. However, Rad51 is expressed in both mitotic and meiotic cells, but Dmc1 is a meiosis-specific recombinase. The underlying mechanism of this differential requirement is unclear. Here, we utilized single-molecule tethered particle motion experiments to compare the kinetics of nucleoprotein filament assembly of Saccharomyces cerevisiae Rad51 and Dmc1. Nucleation on single-stranded DNA (ssDNA) is the rate-limiting step of the nucleoprotein filament assembly. We found distinct differences of these two recombinases: (1) ScRad51 and ScDmc1 have different nucleation preferences of DNA structures. (2) The nucleation rate of ScRad51 is much faster than ScDmc1, indicating that ScRad51 has better ssDNA binding affinity, and preferentially assembles on ssDNA. However, ScDmc1 preferentially nucleates on DNA substrates with duplex DNA/ssDNA junction containing a 3′-ssDNA overhang, as it allows filament extension from 5′-to-3′ direction. Same 5′ ds/ssDNA junction preference is also found in mouse DMC1, suggesting the general and important role of this nucleation site preference for the Dmc1 recombinase. Surprisingly, in the DNA substrates containing short discontinuous patches of ScRad51, ScDmc1 assembly is stimulated. Our data imply that the nucleation of ScDmc1 on ssDNA requires docking sites, such as duplex DNA/ssDNA junction or Rad51 binding on ssDNA. Higher ssDNA affinity of ScRad51 might offer the nucleation docking sites for ScDmc1 assembly during meiosis.