Добірка наукової літератури з теми "Paralogues de Rad51"

The efficient repair of double–strand breaks in DNA is critical for the maintenance of genome stability. In response to ionizing radiation and other DNA–damaging agents, the RAD51 protein, which is essential for homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualized by microscopy and are thought to represent sites where repair reactions take place. The formation of RAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins known as the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that the components present within foci assemble in a carefully orchestrated and ordered manner. By contrast, RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the need for BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 through a series of degenerative motifs known as the BRC repeats. These interactions modulate the ability of RAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role in controlling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNA binding) level.

In this review we focus on new insights that challenge our understanding of homologous recombination (HR) and Rad51 regulation. Recent advances using high-resolution microscopy and single molecule techniques have broadened our knowledge of Rad51 filament formation and strand invasion at double-strand break (DSB) sites and at replication forks, which are one of most physiologically relevant forms of HR from yeast to humans. Rad51 filament formation and strand invasion is regulated by many mediator proteins such as the Rad51 paralogues and the Shu complex, consisting of a Shu2/SWS1 family member and additional Rad51 paralogues. Importantly, a novel RAD51 paralogue was discovered in Caenorhabditis elegans, and its in vitro characterization has demonstrated a new function for the worm RAD51 paralogues during HR. Conservation of the human RAD51 paralogues function during HR and repair of replicative damage demonstrate how the RAD51 mediators play a critical role in human health and genomic integrity. Together, these new findings provide a framework for understanding RAD51 and its mediators in DNA repair during multiple cellular contexts.

RAD51 is involved in finding and invading homologous DNA sequences for accurate homologous recombination (HR). Its paralogs have evolved to regulate and promote RAD51 functions. The efficient gene targeting and high HR rates are unique in plants only in the moss Physcomitrium patens (P. patens). In addition to two functionally equivalent RAD51 genes (RAD1-1 and RAD51-2), other RAD51 paralogues were also identified in P. patens. For elucidation of RAD51’s involvement during DSB repair, two knockout lines were constructed, one mutated in both RAD51 genes (Pprad51-1-2) and the second with mutated RAD51B gene (Pprad51B). Both lines are equally hypersensitive to bleomycin, in contrast to their very different DSB repair efficiency. Whereas DSB repair in Pprad51-1-2 is even faster than in WT, in Pprad51B, it is slow, particularly during the second phase of repair kinetic. We interpret these results as PpRAD51-1 and -2 being true functional homologs of ancestral RAD51 involved in the homology search during HR. Absence of RAD51 redirects DSB repair to the fast NHEJ pathway and leads to a reduced 5S and 18S rDNA copy number. The exact role of the RAD51B paralog remains unclear, though it is important in damage recognition and orchestrating HR response.

The RADiation sensitive protein 51 (RAD51) recombinase is a eukaryotic homologue of the bacterial Recombinase A (RecA). It is required for homologous recombination of DNA during meiosis where it plays a role in processes such as homology searching and strand invasion. RAD51 is well conserved in eukaryotes with as many as four paralogues identified in vertebrates and some higher plants. Here we report the isolation and preliminary characterisation of four RAD51 gene family members in hexaploid (bread) wheat (Triticum aestivum L.). RAD51A1, RAD51A2 and RAD51D were located on chromosome group 7, and RAD51C was on chromosome group 2. Q-PCR gene expression profiling revealed that RAD51A1 was upregulated during meiosis with lower expression levels seen in mitotic tissue, and bioinformatics analysis demonstrated the evolutionary linkages of this gene family to other eukaryotic RAD51 sequences. Western blot analysis of heterologously expressed RAD51 from bread wheat has shown that it is detectable using anti-human RAD51 antibodies and that molecular modelling of the same protein revealed structural conservation when compared with yeast, human, Arabidopsis and maize RAD51A orthologues. This report has widened the knowledge base of this important protein family in plants, and highlighted the high level of structural conservation among RAD51 proteins from various species.

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

The RAD51 paralogues act in the homologous recombination (HR) pathway of DNA repair. Human RAD51C (hRAD51C) participates in branch migration and Holliday junction resolution and thus is important for processing HR intermediates late in the DNA repair process. Evidence for early involvement of RAD51 during DNA repair also exists, but its function in this context is not understood. In this study, we demonstrate that RAD51C accumulates at DNA damage sites concomitantly with the RAD51 recombinase and is retained after RAD51 disassembly, which is consistent with both an early and a late function for RAD51C. RAD51C recruitment depends on ataxia telangiectasia mutated, NBS1, and replication protein A, indicating it functions after DNA end resection but before RAD51 assembly. Furthermore, we find that RAD51C is required for activation of the checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage. This suggests that hRAD51C contributes to the protection of genome integrity by transducing DNA damage signals in addition to engaging the HR machinery.

BackgroundMeiotic homologous recombination (HR) plays an essential role in gametogenesis. In most eukaryotes, meiotic HR is mediated by two recombinase systems: ubiquitous RAD51 and meiosis-specific DMC1. In the RAD51-mediated HR system, RAD51 and five RAD51 paralogues are essential for normal RAD51 function, but the role of RAD51 in human meiosis is unclear. The knockout of Rad51 or any Rad51 paralogue in mice exhibits embryonic lethality. We investigated a family with meiotic arrest, azoospermia and infertility but without other abnormalities.MethodsHomozygosity mapping and whole-exome sequencing were performed in a consanguineous family. An animal model carrying a related mutation was created by using a CRISPR/Cas9 system.ResultsWe identified a 1 bp homozygous substitution (c.41T>C/p.Leu14Pro) on a RAD51 paralogue, namely, XRCC2, in the consanguineous family. We did not detect any XRCC2 recessive mutation in a cohort of 127 males with non-obstructive-azoospermia. Knockin mice with Xrcc2-c.T41C/p.Leu14Pro mutation were generated successfully by the CRISPR/Cas9 method. The homozygotes survived and exhibited meiotic arrest, azoospermia, premature ovarian failure and infertility.ConclusionA XRCC2 recessive mutation causing meiotic arrest and infertility in humans was duplicated with knockin mice. Our results revealed a new Mendelian hereditary entity and provided an experimental model of RAD51-HR gene defect in mammalian meiosis.

Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55–Rad57 in 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

Trypanosoma brucei evades host acquired immunity by antigenic variation, involving periodic switches in the variant surface glycoprotein (VSG) coat. DNA recombination is critical in this process and a key component of homologous recombination, RAD51, also plays a role in VSG switching. T. brucei encodes four proteins distantly related to RAD51; termed RAD51 paralogues, named RAD51-3, RAD51-4, RAD51-5 and RAD51-6. Two of these RAD51 paralogues, RAD51-3 and RAD51-5, have been shown to function in homologous recombination, DNA repair and RAD51 re-localization into foci following DNA damage. Surprisingly, however, only RAD51-3 appears to act in VSG switching. To examine the functions of all the RAD51 paralogues in T. brucei, reverse genetics has been used to generate mutants of the two remaining unstudied paralogues, RAD51-4 and RAD51-6. Phenotypic analysis of both mutant cell lines indicates that these factors also play critical roles in RAD51-directed recombination and repair, and both influence VSG switching in the parasite. As homozygous mutant cell lines of RAD51 and the RAD51 paralogues were available, it was possible to comprehensively compare the phenotypes of rad51 -/- with rad51-3 -/-, rad51-4 -/-, rad51-5 -/-, and rad51-6 -/-. From these results it was observed that the phenotypes of the rad51 paralogue mutants are broadly equivalent, with two exceptions. Firstly, as mentioned above, RAD51-5 does not function in VSG switching, and RAD51-4 and RAD51-6 may not have direct roles in VSG switching. Secondly, rad51-4 -/- mutants are less sensitive to the DNA damaging agent phleomycin and a higher percentage of rad51-4 -/- cells form DNA damage-induced RAD51 foci compared with the other homozygous mutant cell lines. These results may imply that RAD51-4 and RAD51-5 have a less central role in RAD51-directed DNA repair or that their functions can be performed by other factors. In addition, the physical interactions of all the RAD51 paralogues were examined. It was found by yeast two-hybrid and co-immunoprecipitation that they form at least two complexes, and probably function in sub-complexes in a similar manner to the Rad51 paralogues of higher eukaryotes. These analyses shed light on the evolution and role of eukaryotic RAD51 paralogues in DNA recombination and repair in general, as well as the contribution that recombination makes to antigenic variation in T. brucei.

La recombinaison homologue (RH) est un processus fondamental conservé chez les tous les organismes vivants. Chez les mammifères, ce processus implique de nombreuses protéines parmi lesquelles Rad51 et ses paralogues. Afin de déterminer si ces paralogues agissent dans la même voie que RAD51, nous avons réalisé une étude d'épistasie en associant la sur-expression d'un dominant négatif de RAD51 avec une mutation dans un des paralogues, le gène XRCC2. Cette étude montre que les protéines Xrcc2 et Rad51 sont impliquées dans la même voie pour la résistance aux stress génotoxiques, la recombinaison homologues et la duplication des centrosomes. Nous avons également montré que les lignées défectives pour la RH présentent spontanément un ralentissement de la vitesse de progression des fourches de réplication et l'activation d'un « checkpoint » S/G2 dépendant d'ATM/ATR
Homologous recombination (HR) is a fundamental biological process, conserved in all organisms. In mammals, Rad51 protein and its paralogues are involved in this process. Here, we address the question whether RAD51 and its paralogs act in the same pathway. To answer this question, we examined the consequences of the overexpression of a dominant negative form of RAD51 in the irs 1 cell line, mutated in the XRCC2 paralogue gene. This work demonstrated that Rad51 and Xrcc2 proteins act in the same. . Pathway with respect to résistance to genotoxic stresses, homologous recombination and centrosome duplication. In cell lines defective for HR, we also observed a slowing down in the progression of replication forks and the activation of S/G2 checkpoint dependent on ATM/ATR

O Glioblastoma Multiforme (GBM) é o tipo de tumor cerebral maligno com maior incidência na população. A perda do gene PTEN (fosfatase e tensina homóloga) é uma alteração comum associada ao GBM (até 60%) e esse gene codifica uma enzima que antagoniza a ação de PI3K, inibindo a fosforilação de AKT e, desse modo, regulando vias de sinalização relativas à sobrevivência celular e proliferação. Mutações em PTEN têm sido associadas à instabilidade genômica e ao aumento no número de quebras de fita dupla, além de serem relacionadas também à redução da expressão de RAD51, a qual é uma proteína-chave da via de reparo por recombinação homóloga (HR). Diante disso, o objetivo deste estudo foi avaliar se o status de PTEN interfere na expressão de RAD51 e proteínas parálogas (RAD51C e RAD51B) e, consequentemente, se PTEN é capaz de influenciar a eficiência de HR. Com o objetivo de induzir a formação de quebras de fita duplas (DSBs) no DNA, as células foram tratadas com a droga antitumoral etoposídeo, que produz quebras no DNA, principalmente duplas (DSBs). Duas linhagens de GBM com status diferentes de PTEN foram utilizadas: T98G (PTEN mutado) e LN18 (PTEN tipo selvagem). As células de GBM foram tratadas com etoposídeo em diferentes experimentos ou ensaios: proliferação celular, quantificação da necrose e apoptose, cinética do ciclo celular, imunofluorescência da proteína ?- H2AX, quantificação dos níveis de expressão de RAD51 e parálogas e o silenciamento de PTEN na linhagem LN18. Os resultados mostraram que a linhagem LN18 foi mais sensível à droga nos tempos iniciais (24 e 72 h) (até 61,2% de redução), em comparação com a T98G (até 12,3% de redução); no tempo mais tardio de análise (120 h), ambas as linhagens sofreram redução acentuadana proliferação. Adicionalmente, a LN18 exibiu maior porcentagem de células apoptóticas e necróticas, em comparação com a linhagem T98G, nos tempos de24, 72 e 120 horas após o tratamento. O ensaio de imunofluorescência revelou maior indução de células positivas para ?-H2AX na linhagem LN18 em relação à T98G (p =<0,001), após tratamento com etoposídeo (50 e 75 ?M). Nessas concentrações, a análise da cinética do ciclo celular mostrou um bloqueio na fase G2 em ambas as linhagens (p<0,01) nos tempos analisados (24, 48 e 72h), mas apenas a linhagem LN18 revelou bloqueio na fase S. A expressão de RAD51, RAD51B e C foi mais elevada em LN18 em comparação com a T98G e U87MG, nas células tratados (75?M) e controles. PTEN foi silenciado (siRNA-PTEN) na linhagem LN18 para verificar se a redução da expressão desse gene reduziria também a expressão de RAD51 e parálogas. Após 72 horas de silenciamento, com 69,9% de inibição de PTEN, a expressão de RAD51 e RAD51C também se mostrou reduzida em relação ao grupo controle. Em conjunto, os resultados obtidos no presente estudo indicam que o status de PTEN é crucial para as vias de sobrevivência, controle do ciclo celular e indução de apoptose nas células de GBM, indicando a relação entre PTEN e RAD51 e parálogas nas células de GBM tratadas com um indutor de quebras no DNA. Adicionalmente, outras ferramentas de estudo são requeridas para investigar as vias moleculares e possíveis interações e complexos proteicos envolvendo a participação de PTEN e RAD51 e suas proteínas parálogas
Glioblastoma multiforme (GBM) is the most common malignant brain tumor. Loss of PTEN (Phosphatase and tensin homolog deleted on chromosome 10) gene is the most frequent alteration associated with GBM and encodes a phosphatase enzyme that antagonizes the PI3K, by inhibiting AKT phosphorylation thereby regulating signaling pathways related to cell survival and proliferation. PTEN deficiency has been associated with genomic instability and increased endogenous DSBs, as well as reduced expression of RAD51, which is a key gene with crucial role in HR. In this study, we aimed to evaluate whether the PTEN status in GBM cell lines can affect RAD51 expression and HR efficiency under conditions of treatment with the antineoplastic drug etoposide, which targets the DNA topoisomerase II enzyme, thus leading to the production of DNA breaks. T98G (PTEN mutated) and LN18 (PTEN wild-type) cells were treated with etoposide, and several assays were carried out: cell proliferation, detection and quantification of necrosis and apoptosis, cell cycle kinetics, immunofluorescence staining, RAD51 (and paralogs) protein expression, and PTEN silencing in LN18 cell line, by using the siRNA method. LN18 cells showed a greater reduction in cell proliferation, compared to T98G after treatments (25, 50, 75 e 100 µM) at 24, 72 and 120h. Both cell lines showed a significant increase (p=<0.001) in cell death induction, but LN18 presented a greater percentage of apoptotic and necrotic cells than T98G (24, 72 and 120h). The induction of DSB was analyzed by immunostaining (with ?-H2AX antibody), and for the concentrations (50 and 75 µM) tested, LN18 showed higher levels of ?-H2AX positive cells than that observed for T98G (p=<0.001). The analysis of cell cycle kinetics performed for cells treated with etoposide (50 and 75 µM) and collected at 24, 48 and 72h, LN18 presented a greater G2-blockage, as compared to T98G; only LN18 showed a blockage at the S-phase. The expression of RAD51, RAD51B and C was higher in LN18 compared to T98G and U87MG cells treated with etoposide (75 µM) and controls. When we silenced PTEN in LN18 linage, to check if PTEN silencing may reduce the expression of RAD51 and its paralogs, we found a 69.9% reduction in PTEN protein expressions, and the expression of RAD51 and RAD51C was also found reduced, compared to the control group. Taken together, the results obtained in this study indicate that the status of PTEN is critical for survival pathways, cell cycle control and induction of apoptosis in GBM cells, confirming the relationship between PTEN and RAD51 and its paralogs in GBM cells treated with an inducer of DNA breaks. These results contribute with relevant information for further studies on molecular pathways underlying the interaction between PTEN and RAD51 and its paralogs

Homologous recombination (HR) is an essential DNA break repair mechanism that remains incompletely understood. HR is a complex multistep process initiated by the loading of RAD-51 recombinase as filaments onto single stranded DNA (ssDNA). This structure directly invades an intact homologous duplex, which serves as a template for repair DNA synthesis. Numerous positive regulators of HR have been described, including the Rad51 paralogs, but the mechanism of action of Rad51 paralogs in promoting HR is unknown. In this study, I have characterized the mechanism of action of a novel Rad51 paralog complex, RFS-1/RIP-1, from C. elegans. RFS-1 is a Rad51 paralog required for RAD-51 focus formation at stalled replication forks, indicating an early positive regulatory role in HR. I demonstrate that RFS-1 interacts with a nematode-specific orphan protein, RIP-1. I identify a cryptic Walker B ATPase-like motif within RIP-1, which is functionally important in establishing the RFS-1/RIP-1 interaction interface. rip-1 and rfs-1 mutant animals phenocopy for essentially all phenotypes analysed. Together these data suggest RFS-1/RIP-1 functions as a constitutive complex. I show recombinant RFS-1/RIP-1 can be purified and specifically binds ssDNA but lacks measurable ATPase activity. RFS-1/RIP-1 also strongly stimulates strand invasion activity by RAD-51, consistent with a pro-recombinogenic function in vivo. I define for the first time the mechanism of action underlying the intrinsic ability of Rad51 paralogs to stimulate HR. Using a combination of biochemical and biophysical approaches, notably electrophoretic mobility shift assays, stopped-flow reaction kinetics and nuclease protection assays, I show RFS-1/RIP-1 dramatically alters the properties of RAD-51-ssDNA filaments such that RAD-51 is more stably associated with ssDNA yet the ssDNA is more sensitive to nuclease degradation. RFS-1/RIP-1 exerts these effects primarily downstream of filament formation, ruling out a major role in RAD-51 loading. I propose RFS-1/RIP-1 remodels RAD-51-ssDNA filaments to a conformation poised for pairing with the template duplex and strand invasion.

Repair of DNA damage is one of the most important processes undergone in a dividing cell. This is a two-part study undertaken to better understand some of the proteins involved in the sensing and repair of DNA damage in Drosophila melanogaster. The first portion of this experiment followed two Drosophila Rad51 paralogs, dmRad51D and dmXRRC2, and using constructs tagged with GFP, found that they entered the nucleus in response to drug induced DNA damage. Approximately one hour after the induction of DNA damage via bleomycin, dmRad51D and dmXRCC2 entered the nucleus of the Drosophila culture cells, where they remained for the next three to four hours. Following this period in the nucleus, the cells were visualized moving back into the cytosol. The second portion of this experiment was concerned with the four Drosophila Rad51 paralogs (dmRad51 D, dmXRCC2, Spn B, and Spn D) and two paralogs from Homo sapiens (hsRad51 D and dmRad51 D) and their interactions.

The integrity of all genomes is under constant threat, with DNA double strand breaks being particularly dangerous. Double strand breaks can be repaired by homologous recombination, a process catalysed by recombinase proteins of the RecA family. The archaeal recombinase, RadA, is homologous to eukaryotic and bacterial Rad51/RecA. Euryarchaea encode an additional Rad51/RecA homologue, RadB. RadB shares homology with the core domain of RadA and has been shown to bind both single and double stranded DNA, binds ATP and possesses a very weak ATPase activity. However, RadB does not catalyse strand exchange. RadB has been shown to interact with RadA, a Holliday junction resolvase (Hjc) and a DNA polymerase (PolD), suggesting a role in recombination. In this study, radB was deleted from the halophilic archaeon, Haloferax volcanii. 'delta' radB strains were slow growing, sensitive to mitomycin C and UV irradiation, and deficient for both crossover and non-crossover recombination. Deletion of radA results in similar phenotypic characteristics, and complete abrogation of recombination. Strains deleted for both radA and radB are equally defective as 'delta' radA strains, demonstrating that RadA is epistatic to RadB. A suppressor of 'delta' radB was isolated and identified as a mutation in the polymerisation domain of RadA (RadA-A196V). radA-A196V suppresses the slow growth, crossover and non-crossover recombination defects associated with 'delta' radB, as well as UV and mitomycin C sensitivity phenotypes. On account of the nature of this suppressor, the observed interaction between RadA and RadB, and the epistatic relationship between RadA and RadB, a role for RadB as a recombination mediator protein is proposed. Finally, strains were deleted for hjc. 'delta' hjc strains exhibit no growth, crossover and non-crossover recombination defects and no UV and mitomycin C sensitivity. This suggests that another, as yet unidentified, Holliday junction resolvase is encoded by Haloferax volcanii.

Acquisition of invasiveness through extracellular matrix is a crucial characteristic of transition to malignancy in the breast. It was previously shown that Polo-like kinase 1 (PLK-1), a mitotic kinase and genome stability regulator, is involved in acquisition of invasiveness in a breast cell model (HMT-3522 cell line) of pre-invasive to invasive transition. This and other data led to the suggestion that a new class of genes called GISEM for Genome Instability and Extracellular Matrix Invasiveness may exist. Previous lab data show that XRCC3 is found downregulated in progression from preinvasive to invasive phenotype. This led to the hypothesis that XRCC3 may be a negative regulator of invasion. To support this hypothesis, overexpression of XRCC3 in the invasive T4-2 cells downregulated invasion, but also growth. In order to verify the role of XRCC3 in invasiveness, and determine whether it is independent from any effects on growth, we tested the effect of downregulating XRCC3 on the invasiveness of T4-2 cells. Short-term downregulation of XRCC3 using siRNAs produced a significant increase in invasiveness, suggesting a role for XRCC3 as a negative regulator of invasion. During the invasion assay time course, XRCC3 downregulation had no effect on growth or apoptosis supporting the idea that this is a direct effect on invasion and not an artifact of the assay. XRCC3 is one amongst the five members of the RAD51 paralog family, consisting of accessory proteins or RAD51 cofactors (namely RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) which interact with each other to form complexes (BCDX2, BC, DX2 and CX3) that collaboratively assist RAD51 in homologous recombinational repair (HRR) of DNA double-strand breaks. To see if these interactions are important in terms of invasion, as they have been demonstrated for DNA repair, we studied the effect of XRCC3 downregulation on the levels of RAD51 paralogs. We found lowered levels of RAD51C, but not RAD51B or RAD51D, when XRCC3 was downregulated. Since XRCC3 forms the CX3 complex with RAD51C, we downregulated RAD51C using siRNAs in T4-2 cells and found this to significantly increase invasiveness. Consistent with previous findings by other groups, downregulating RAD51C also lead to decreased levels of XRCC3 in invasive T4-2 cells. These results suggest that the XRCC3-RAD51C interaction is important for invasion as well as the previously studied DNA repair function. In delineating the mechanism by which XRCC3 acts as a negative regulator of invasion, we further questioned if XRCC3 alters secreted factors that are important for the invasiveness of T4-2 cells and tested the effects of conditioned medium (CM) from XRCC3 altered T4-2 cells on parental T4-2 cells’ ability to invade. Results show a significant increase in invading T4-2 cells when suspended in CM from XRCC3 siRNA transfected T4-2 cells, suggesting a direct effect of XRCC3 siRNAs on the ability of T4-2 CM to induce invasiveness in T4-2 cells. Furthermore, we investigated the effects of XRCC3 inhibition on cell surface integrins and focal adhesion kinase (FAK). Indirect immunofluorescence results show increased formation of focal adhesions containing two phosphorylated FAK residues- autophosphorylated FAK-Y397 and FAK-Y861 (previously implicated in increased migration and invasion of tumor cells) in XRCC3 siRNA transfected T4-2 cells. Overall, these results support a new role of XRCC3 in invasion, in addition to its previously reported role in DNA repair. These findings imply that loss of XRCC3 function in cancer progression would upregulate invasion as well as downregulate DNA repair and genome stability. Therefore, stabilization of XRCC3 function could provide a promising therapeutic against breast cancer progression. The dual role of XRCC3 in invasion and DNA repair also renders it an attractive candidate risk biomarker of breast pre-cancer to invasive cancer progression.