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.

There are multiple mechanisms by which DNA can become damaged. Such damage must be repaired for the cell to avoid ill-health consequences. Homologous recombination (HR) is a means of repairing one specific type of damage, a double-strand break (DSB). This complex pathway includes the Rad51-DNA nucleoprotein filament as its primary machinery. Current methodology for studying HR proteins includes the use of fluorescently labeled DNA to probe for HR dynamics. This technique limits the number of proteins that can be involved in experimentation, and often only works as an end reporter. The work here aims at improving upon standard techniques by creating two fluorescent protein probes. The first probe was developed by directly attaching a fluorophore to Saccharomyces cerevisiae Rad51 with the use of click chemistry and the incorporation of unnatural amino acids. This probe could function as a primary reporter on the formation and dissociation of the Rad51-DNA filament in the presence of pro- and anti- HR mediator proteins. The second probe was created by labeling the exterior cysteine residues of Plasmodium falciparum single strand DNA binding protein (SSB) with a fluorophore via maleimide chemistry. This probe acts as a secondary reporter for HR dynamics by signaling for when free single stranded DNA (ssDNA) is available.

Dans cette thèse, nous avons étudié les aspects mécanique et thermodynamique de la recombinaison homologue, un processus crucial de réparation de l'ADN. Des travaux préliminaires d'observation de molécules d'ADN étirées lors de la recombinaison homologue ont mis à jour la complexité de ce processus et la difficulté de l'observer en fluorescence. Nous avons ensuite développé une nouvelle technique appelée "pinces magnétiques sensibles au couple" nous permettant de maintenir une molécule unique d'ADN avec une force et un couple connus tout en mesurant son état de torsion avec une résolution de quelques degrés. Nous avons ainsi montré de manière directe que la polymérisation de la recombinase hRad51 a lieu par ajout de monomères chacun déroulant l'ADN de 65° en moyenne. Nous avons également été capables de mesurer le couple d'arrêt de la polymérisation. Une modélisation mécano-chimique nous a finalement permis d'évaluer le potentiel chimique de la polymérisation, en bon accord avec les données biochimiques existantes.

In this study, the role of two DNA repair genes, X-ray repair cross complementing group 3 (XRCC3) Thr241Met and Rad51 G135C polymorphisms were investigated in the risk of development of childhood ALL in Turkish population among 193 healthy controls and 184 ALL patients, by using PCR-RFLP technique. For XRCC3 Thr241Met polymorphism, the frequencies of both heterozygous and homozygous mutant genotypes were found to be higher in the controls compared to ALL patients (OR: 0.59, p = 0.02 / OR: 0.48, p = 0.02, respectively). In addition, either heterozygous (Thr/Met) or homozygous mutant (Met/Met) genotypes were significantly more common in the controls than the ALL patients (OR: 0.55, p =0.005). In case of Rad51 G135C polymorphism, no significant associations have been found with the risk of childhood ALL. Combination of XRCC3 heterozygote and Rad51 heterozygote genotypes increased the protective effect for risk of childhood ALL. (OR=0.35 / p =0.02). Combination of homozygote mutant genotype of XRCC3 with homozygote wild type genotype of Rad51 gave a highly statistically proved protective effect for the development of disease (OR= 0.36 / p= 0.004). To our knowledge, this is the first study showing the protective role of XRCC3 Thr241Met polymorphism either alone or in combination with Rad51 G135C variant on the risk of development of childhood ALL. In addition, interactions of these polymorphisms with non-genetic risk factors were investigated. Only in terms of paternal exposure, the heterozygote (Thr/Met) genotype for XRCC3 gene in children whose father exposed to cigarette smoke demonstrated a significant risk of 3.0 fold (p=0.05). Moreover, the frequency of Rad51 135C allele was determined for the first time in Turkish population. The frequency of the mutant allele was found to be very similar to that observed in other Caucasian populations.

DNA damage occurs through endogenous and exogenous sources, and can lead to stalled replication forks, genetic disorders, cancer, and cell death. Homologous recombination (HR) is a relatively fast and error-free repair pathway for damaged DNA, which can occur through a gene conversion event or through a crossing-over event with the exchange of genetic material. Homologous recombination occurs most frequently in the G2 phase of the cell cycle and utilizes the sister chromatid as the repair template. When the sister chromatid is unavailable, the homologous chromosome or a homologous sequence in an ectopic location can be used to repair the lesion; the latter of which is referred to as ectopic homologous recombination (EHR). Rad51 is a key protein involved in HR, and to test its role in EHR, variant Rad51 proteins were expressed in murine hybridoma cells. These Rad51 variants were assayed for their effects on EHR. Excess wild-type Rad51 as well as a deficiency of wild-type Rad51 decreased EHR from the background level found in these cell lines. Thus, Rad51 is necessary for EHR, but there may be an optimal amount of Rad51 required for efficient EHR. Expression of the Rad51 catalytic mutants Rad51K133A and Rad51K133R was found to have an inhibitory effect on EHR, as expected based on the loss of ATP binding and ATP hydrolysis, respectively, in these variants. Excess wild-type Rad51 was verified in this study to increase HR via a gene targeting assay. MMC treatment, but not ionizing radiation, leads to an increase in EHR in the presence of excess wild-type Rad51. Thus, endogenous levels of Rad51 are sufficient to maintain EHR, but in the presence of excess wild-type Rad51, the level of EHR can increase in response to certain DNA damaging agents and in response to gene targeting.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Dissertation (Ph.D.)--University of Toledo, 2008. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: pages 73-77, 93-95, 109-111, 145-172.

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anaemia (FA) gene, following the discovery of two patients carrying dominant negative mutations. FA is a hereditary DNA repair disorder characterised by various congenital abnormalities, progressive bone marrow failure and cancer predisposition. The cellular and molecular pathology of FA is poorly understood, resulting in a severe lack of effective treatment options. In this thesis, I describe the first viable vertebrate model of RAD51 loss. Phenotypic characterisation of zebrafish rad51 loss-of-function mutants showed that they develop key features of FA, including hypocellular kidney marrow, sensitivity to crosslinking agents and decreased size. Taking advantage of the unique properties of the zebrafish model, I show that some of these symptoms stem from both decreased proliferation, as well as increased apoptosis of embryonic haematopoietic stem and progenitor cells. Co-mutation of p$L was able to rescue the haematopoietic defects seen in the single mutants, but led to tumour development, underscoring the role of rad51 as a tumour suppressor. I further demonstrate that prolonged inflammatory stress can exacerbate the haematological impairment, leading to an additional decrease in kidney marrow cell numbers. In contrast, prolonged aldehyde-derived stress did not induce symptoms in the mutant fish. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signalling during embryogenesis leads to the haematological defects seen later in life in FA. It also strengthens the evidence for the involvement of haematopoietic stress, such as inflammation, in the development of bone marrow failure. Further research on this novel zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and check-point activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

The Role of TvTrxR in Drug Resistance and Characterization of TvRad51 in Homologous Recombination in Trichomonas vaginalis Abstract By Melissa Hopper University of the Pacific 2016 In recent years, prevalence of metronidazole-resistant cases of Trichomonas vaginalis has been on the rise. With nearly 10% of strains resistant to metronidazole, new treatments to combat this parasite have become a necessity. FDA-approved drug screens have identified the compound, auranofin, as an effective agent against similar protozoans. The mechanism of inhibition by auranofin has been found to proceed through inhibition of the thioredoxin-based anti-oxidant pathway, targeting the enzyme thioredoxin reductase (TrxR). In this study, auranofin was found to be an effective inhibitor of T. vaginalis TrxR activity. Auranofin was also found to be an effective inhibitor of several trichomonad strains in culture, exhibiting IC50 values comparable to metronidazole. These studies indicate that auranofin is a promising agent for treatment of trichomoniasis. Another aspect of T. vaginalis biology addressed in this study is the ability of T. vaginalis to carry out homologous recombination (HR), a process used to repair double-stranded breaks in DNA. The protein radiation sensitive protein 51 (Rad51) plays a crucial role in the process of HR in mitotic and meiotic recombination. In this study, experiments were carried out to elucidate the role of T. vaginalis Rad51 in homologous recombination. TvRad51 was found to exhibit nuclear localization and was capable of carrying out ATP hydrolysis. Rad51 was shown to be up-regulated at the protein level in T. vaginalis in response to treatment with DNA-damaging agents. In addition, TvRad51 was capable of binding the BRC repeat region of TvBRCA2. These results indicate that T. vaginalis upregulates expression of Rad51 protein in response to certain forms of DNA damage and TvRad51 may be capable of carrying out HR mediated by different binding partners.