Добірка наукової літератури з теми "Rad51 filament"

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.

The accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions; however, they can be repaired using homologous recombination. Homologous recombination is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, which is a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, which are collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here, we discuss the current perspective on the in vivo and in vitro function of the RAD51 paralogs, and their relationship with cancer in vertebrate models.

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.

ABSTRACT A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1 − mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.

ABSTRACT The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. We suggest the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding.

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.

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.

Abstract We showed before that cells transformed by BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2 and TEL/PDGFR, inducing chronic myeloproliferative disorders (MPDs), and CD34+ chronic myeloid leukemia (CML) stem/ progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain an excess of DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress [Blood, 2005; Cell Cycle, 2006; DNA Repair, 2006; Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cellenriched populations seem to display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Elevated levels of DSBs were also observed in leukemia cells expressing imatinib-resistant BCR/ABL kinase mutants. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. Numerous ROS- and radiation- induced DSBs are not lethal for BCR/ABL-positive leukemia cells; instead, they induce chromosomal instability implicating enhanced, but unfaithful repair [Leukemia, 2008]. The previous report [Mol. Cell, 2001] and ongoing studies demonstrated that BCR/ABL kinase (non-mutated and imatinib-resistant mutants) modulates expression of the mammalian RecA homologs RAD51, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which are responsible for homologous recombination repair (HRR) of DSBs. RAD51 plays a key role in HRR in cells transformed by BCR/ ABL and other FTKs [Mol. Cell, 2001; Mol. Cell. Biol., 2002]. BCR/ABL stimulates the expression of, interacts with and phosphorylates RAD51 on Y315, which is located in a critical fragment of RAD51 essential for its filament formation on DNA. Accordingly, our recent results indicated that BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for nuclear RAD51 foci formation in response to DNA damage. In addition to RAD51, BCR/ABL interacts directly with and phosphorylates RAD51B and XRCC2, but not other RecA homologs. Altogether, it appears that BCR/ABL can deregulate the expression and phosphorylation of some RecA homologs, which may have a significant impact on the efficiency and fidelity of DSB repair resulting in protection from apoptosis and chromosomal instability. Therefore, disassembly of BCR/ABL from RecA homologs should reduce the capability of CML cells to repair numerous ROS-induced DSBs and eventually trigger apoptosis. Based on this hypothesis we investigated the mechanisms of association between BCR/ABL and RecA homologs. Interactions between BCR/ ABL and RAD51 or RAD51B depend on the proline- rich (PP) regions of RAD51 and RAD51B, and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL, which form a pocket binding the PP regions. Disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) abrogated direct interaction with the BCR/ABL SH3-SH2-CD pocket. On the other hand, single amino acid substitutions in the BCR/ABL SH3-SH2-CD pocket, which eliminated its capability of binding the PP regions, prevented complex formation with RAD51 and RAD51B. In addition, RAD51 and RAD51B may interact with members of the BCR/ABL proteome such as Grb2 and Shc (RAD51 and RAD51B), and c-CrkL (only RAD51B), but not Gab2 and c-Cbl. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3-SH2-CD pocket mutant, where single amino acid substitutions disrupted its direct interaction with RAD51, displayed a slower proliferation rate and responded poorly to genotoxic stress despite intact kinase activity in comparison to cells transformed with non-mutated BCR/ABL. Interestingly, expression of RAD51 PP-LL mutant eliminated BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. These results suggest that the interaction between BCR/ABL and RAD51 may be targeted for selective elimination of leukemia cells and/or suppression of genomic instability. To test this hypothesis we are employing the peptide aptamer strategy targeting RAD51 PP regions in CD34+ cells obtained from imatinib-sensitive and imatinib-resistant CML patients and healthy volunteers in vivo and in vitro. In summary, we hypothesize that mechanisms regulating the association of BCR/ABL with RAD51 and other mammalian RecA homologs may be explored for the planning of more effective anti-tumor modalities.

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.