La grand majorité des espèces forment très peu de crossovers (CO) par chromosome en méiose, et ce quelle que soit la taille des chromosomes et en dépit de l'excès de précurseurs de recombinaison disponibles. Les mécanismes qui sous-tendent cette limitation restent, pour une grande part, inconnus. Pour identifier des facteurs limitant la formation des CO en méiose, nous avons mis en place un crible permettant d'isoler des mutants formant plus de CO que le sauvage. Chez Arabidopsis thaliana, les mutants présentant un défaut de CO (ex: les mutants zmm) présentent une réduction de fertilité directement observable sur la longueur des fruits. Notre crible repose sur l'idée qu'une mutation augmentant la formation des CO devrait restaurer la fertilité des mutants zmm.Le premier suppresseur identifié nous a permis de montrer que FANCM est un régulateur majeur de la formation des CO méiotiques, en limitant la voie dépendante de MUS81, normalement minoritaire chez Arabidopsis (Crismani et al., 2012). Nous avons ensuite montré que deux des cofacteurs de FANCM au sein de la voie de l'anémie de Fanconi (FA), MHF1 et MHF2, agissent dans la même voie pour limiter les CO. En dehors de ces trois protéines, les autres protéines FA ne sont pas des protéines anti-CO (Girard et al., 2014).Nous avons ensuite identifié FIDGETIN-Like-1 comme une protéine anti-CO agissant en parallèle de FANCM. En effet, si les deux mutations figl1 et fancm alimentent la voie dépendante de MUS81 de formation des CO, leurs effets sont cumulatifs et mènent à une augmentation d'un facteur 6 du nombre CO par méiose, et ce sans affecter la ségrégation des chromosomes. Ce résultat montre que FIGL1 et FANCM agissent à deux étapes indépendantes de la recombinaison, et nos données suggèrent que FIGL1 pourrait agir en amont de FANCM.Ce travail a révélé l'existence d'au moins deux mécanismes de limitation des CO méiotiques, et montre que la fréquence de CO peut être largement augmentée sans que la ségrégation des chromosomes ne soit affectée. / Most species only few meiotic crossovers (COs) per chromosome irrespective of their physical size and despite an excess of recombination precursors. However, the underlying mechanisms constraining CO frequency remain largely unknown. In order to find factors limiting meiotic COs, we performed a genetic screen to find mutants with increased CO frequency. CO-deficient mutants (e.g. zmm) of Arabidopsis thaliana display reduced fertility, easily noticeable by their obvious reduction in fruit length. We designed a screen based on the idea that mutations that increase CO frequency will restore the fertility of the zmm mutants.We showed first identified FANCM as a major anti-CO protein limiting MUS81-dependent COs, a normally minor pathway in Arabidopsis (Crismani et al., 2012). We then showed that two of FANCM's cofactors from the Fanconi anemia pathway of DNA repair, namely MHF1 and MHF2, act along FANCM to limit meiotic crossovers, whereas the other Fanconi proteins do not (Girard:2014).Another mutant revealed FIDGETIN-Like-1 (FIGL1) as an anti-CO factor that acts in parallel to FANCM. While both figl1 and fancm mutations fuels the MUS81-dependent CO pathway, the effect of both mutations is cumulative, leading to a six-fold increase in CO formation, without impairing chromosome segregation. This shows that FIGL1 and FANCM act independently, and our data suggest that FIGL1 could act at an earlier step in the recombination pathway than FANCM.This work reveals that at least two different mechanisms limit meiotic CO number and shows that CO frequency can be largely increased without affecting chromosome distribution at meiosis.

Le but de cette thèse était d’identifier de nouveaux substrats au complexe FANC Core,déficient dans l’Anémie de Fanconi, une pathologie génétique rare. Cette maladie estcaractérisée par un phénotype hétérogène associant une pancytopénie à des malformationscongénitales et une prédisposition accrues aux leucémies myéloïdes aigues.L’anémie de Fanconi est causée par la mutation biallélique dans un des seize gènesFANC. Les protéines produites par ces gènes participent à une même voie moléculaireimpliquée dans la signalisation des dommages de l’ADN. Huit de ces protéines forment lecomplexe FANC Core, une E3 ubiquitine ligase, dont les seuls substrats à ce jour sontFANCD2 et FANCI.Dans le but d’identifier de nouveaux substrats du complexe FANC Core, j’ai réalisé uneanalyse protéomique après immunoprécipitation des peptides modifiés par l’ubiquitine ou parles ubiquitin-like NEDD8 et ISG15. Cette expérience a été faite dans des cellules déficientespour la voie FANC, mutées sur les gène FANCA ou FANCC et comparée à des cellulescorrigées par l’expression de ces gènes.Cette analyse révèle que FANCD2 et FANCI sont les seules cibles du complexe FANCCore en réponse à des dommages de l’ADN.Néanmoins, je montre l’existence d’autres protéines qui sont modifiées d’une manièreFANCA dépendante. Ces protéines sont pour la grande majorité des protéines membranairesou associées aux membranes cytoplasmiques. Parmi celles-ci, j’ai pu déterminer que lerécepteur aux chimiokines, CXCR5, était modifié d’une manière FANCA dépendante parl’ubiquitin-like NEDD8. Cette modification impacte sur la localisation de la protéine à lamembrane et à des conséquences sur la migration des cellules.J’ai aussi montré que FANCA participe d’une manière similaire à la régulation de lalocalisation membranaire d’autres protéines comme APLP2.Ainsi, il est proposé par ce travail un rôle de la protéine FANCA en dehors du complexeFANC Core et en dehors de la réparation des dommages à l’ADN. Comment la protéineFANCA participe à la régulation du trafic des protéines membranaires reste un point nonrésolu à ce jour. / The aim of this thesis was to find new substrates of the E3-ubiquitin ligase activity of theFANC Core complex, mutated in the rare genetic disorder Fanconi Anemia. This disease ischaracterized by bone marrow failure, developmental abnormalities and predisposition tocancer. Eight of the 16 known FANC proteins participate in the FANCcore nuclear complex,which has E3 ubiquitin-ligase activity and monoubiquitinates FANCD2 and FANCI inresponse to replication stress.In this thesis, I used mass spectrometry to compare cellular extracts from FANC Corecomplex deficient FA-A and FA-C cells to their ectopically corrected counterparts after agenotoxic stress.FANCD2 and FANCI appear to be the only true direct targets of the FANCcore complex.However, I also identified other proteins that undergo post-translational modifications throughFANCA- or FANCC-specific direct or indirect mechanisms that are independent of theFANCcore complex. The majority of these potential FANCA or FANCC target proteinslocalize to the cell membrane.Finally, I demonstrated that (a) the chemokine receptor CXCR5 is a neddylated protein; (b)FANCA, surprisingly, appears to modulate CXCR5 neddylation through a currently unknownmechanism; (c) CXCR5 neddylation is involved in the targeting of this receptor to the cellmembrane; and (d) CXCR5 neddylation stimulates cell migration/motility.I also confirmed that the role of FANCA in neddylation is not restrict to CXCR5 but also to,at least, one other protein, APLP2.My work has uncovered a new signaling pathway that is potentially involved in the rarehuman syndrome Fanconi Anemia and in cell motility and has highlighted a potential newfunction for the FANCA protein independant of the FANC Core complex and of a genotoxicstress.

Orientador: Carmen Sílvia Bertuzzo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T12:57:33Z (GMT). No. of bitstreams: 1 Goncalves_ClaudiaEstela_D.pdf: 2258187 bytes, checksum: 7cac2852cc031e31eba1a31c62d9dda2 (MD5) Previous issue date: 2014 / Resumo: A Anemia de Fanconi (AF) é uma alteração genética caracterizada por múltiplas anomalias congênitas, anormalidades hematológicas e predisposição a uma variedade de tumores. A incidência mundial da AF em todo o mundo é de aproximadamente três por milhão e a frequência de heterozigotos é estimada em um para 300 na Europa e Estados Unidos. É uma doença causada por mutações em genes relacionados ao sistema de reparo. Até o momento foram descritos 16 genes que podem estar multados. São eles: FANCA, FANCB, FANCC, FNCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCO, FANCP E PANCQ. Os grupos mais frequentes são o FANCA e FANCC. De qualquer modo devido a essa heterogeneidade gênica, o diagnóstico molecular dessa alteração é complexo. Com o intuito de testar uma estratégia diagnóstica, o presente trabalho se propôs a identificar as mutações mais frequentes no gene FANC por PCR e digestão enzimática e investigar mutações no gene FANCA, por meio da Reação em Cadeia da Polimerase seguida de digestão enzimática da mutação Brasileira e posterior sequenciamento dos 43 éxons em 60 pacientes portadores de Anemia de Fanconi DEB positivos. Foram detectados 19 pacientes (27,94%), como sendo do grupo C e 16 pacientes como grupo A (23,53%). A mutação ?3788-3790 do gene FANCA teve uma frequência alélica de 15,4%. Foram encontradas 3 mutações intrônicas, 1 mutação sinônima e 1 mutação de sentido trocado no gene FANCA. Não foram encontradas correlações com as manifestações hematológicas, renais, baixo peso, malformações congênitas de membros, machas e pigmentação de pele, sexo e idade / Abstract: The Fanconi Anemia (FA) is a genetic disorder characterized by multiple congenital and hematological abnormalities and predisposition to a variety of tumors. The worldwide incidence of AF is approximately three per million and the frequency of heterozygotes is estimated at one in 300 in Europe and the United States. It is a disease caused by mutations in genes involved in the repair system. So far have been described 16 genes that may be mutated. They are: FANCA , FANCB , FANCC , FNCD1 , FANCD2 , FANCE , FANCF , FANCG , FANCI , FANCJ , FANCL , FANCM , FANCN , FANCO , FANCP And PANCQ . The most common groups are the FANCA and FANCC. However due to this genetic heterogeneity, molecular diagnosis of this change is complex. In order to test a diagnostic strategy, the present study aimed to identify the most frequent mutations in the FANC gene by PCR and restriction enzyme digestion and investigate mutations in the FANCA gene, using the polymerase chain reaction followed by enzymatic digestion of the mutation Brazilian and subsequent sequencing of the 43 exons in 60 patients with Fanconi Anemia positive DEB. 19 patients (27.94%) were detected as group C and 16 patients as group A (23.53%). The ?3788 - 3790 mutation in the FANCA gene had an allelic frequency of 15.4%. Three intronic mutations, one synonymous mutation and one mutation changed direction in FANCA gene were found. No correlation with hematologic, renal, low weight manifestations of congenital malformations members, butches and skin pigmentation, age and sex were found / Doutorado / Clinica Medica / Doutora em Clínica Médica

Due to the action of endogenous and exogenous agents, DNA is subject up to 70,000 lesions per day, thus the existence of repair mechanisms and enzymes is more than necessary. We know basic mechanisms of several specific DNA repair pathways, of which the Fanconi anaemia (FA) repair pathway is one of the least explored. FA is a rare, autosomal recessive disorder characterized by early onset bone marrow failure, developmental defects, genomic instability and predisposition to acute myeloid leukaemia and solid tumours. The primary diagnosis of FA is a hypersensitivity to cross-linking agents of DNA due to inactivation of one of the 21 genes from the FA repair pathway, the so-called FANC genes (FA complementation group). The molecular defect in FA is an impaired repair of DNA interstrand cross-links (ICLs). The ICLs are cytotoxic lesions that inhibit the process of DNA replication and transcription. A crucial step in the FA pathway that initiates ICL repair is a monoubiquitination of FANCD2. FANCD2 monoubiquitination is a base for the recruitment of additional proteins that coordinate DNA repair. Ubiquitin is recruited via activating enzyme E1 (UBA1), ubiquitin-conjugating enzyme E2T (UBE2T) and transferred onto FANCD2 by multisubunit E3 ligase (FA core complex). There are up to 11 different proteins...

Fanconi anemia (FA) is a genome instability syndrome that is clinically manifested by bone marrow failure, congenital defects, and elevated cancer susceptibility. The FA pathway is known to regulate the repair of DNA interstrand crosslinks in part through DNA homologous recombination (HR) repair. Up to today 16 FA proteins have been discovered that may participate in the common pathway. Cells that have mutations in the FA genes are hypersensitive to DNA damaging agents and display chromosome instability. A key regulatory event in the FA pathway is monoubiquitination of FANCD2-FANCI heterodimer that is mediated by a multi-component E3 ubiquitin ligase complex called FA core complex. Current model suggests that once the FANCD2-FANCI heterodimer is monoubiquitinated it relocates to chromatin where it interacts with other key repair proteins to facilitate DNA repair. More than 90% of the FA cases are presumed to be associated with defects in the monoubiquitination reaction, suggesting the significance of the modification in the pathogenesis of the disease. Despite the significance, the molecular interplay between the FA core complex and the FANCD2-FANCI heterodimer remains enigmatic. We are interested in the assembly mechanism of the various FA subcomplexes into the core complex, and we are actively investigating how the FANCD2-FANCI heterodimer is recruited to these putative subcomplexes. As the FA pathway is a crucial determinant for cellular resistance to DNA damaging agents, there have been hypotheses that disruption of this pathway may be beneficial in enhancing chemosensitivity of certain cancer cells. In collaboration with Dr. Cai’s chemistry lab, we will develop a screen platform to identify a small molecules to interrupt the monoubiquitination reaction. Completion of these studies will enhance the much-needed knowledge of the key enzymatic reaction in the pathway, and perhaps the information can be used for development of novel chemotherapeutic strategies.

Fanconi anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer predisposition. Cell lines derived from FA patient exhibit chromosomal instability and sensitivity to DNA interstand crosslinkers (ICLs) like mitomycin (MMC). The key event in Fanconi anemia pathway is the regulated ubiquitination and deubiquitination of FANCD2 and FANCI. Upon DNA damage, FANCD2 and FANCI are monoubiquitinated by FA core complex. They then move into the chromatin and serve as the landing site for downstream players, like FANCP/SLX4 and FAN1. USP1, the deubiquitinating enzyme (DUB), removes ubiquitin from FANCD-Ub/FANCI-Ub, and this step is required for the integrity of FA pathway. This dissertation addresses how USP1 is regulated in the cell. In Chapter 2, we discovered UAF1/WDR48 as a critical binding partner for USP1, by activating its enzymatic activity in vitro and in vivo. We then generated DT40 knockout cell lines of USP1 and UAF1. We showed that USP1/UAF1 complex is functionally required for homologous recombination (HR). Interestingly, PCNA-Ub is also a substrate for USP1. We discovered that hELG1, through its binding to USP1/UAF1 complex, regulates the deubiquitination of PCNA-Ub and translesion DNA synthesis (TLS). Then in Chapter 3, we discovered a tandem repeat of SUMO-like domains (SLD1 and SLD2) in the C terminus of UAF1. SLD2 binds directly to a SUMO-like domain-interacting motif (SIM) on FANCI. Deletion of the SLD2 of UAF1 or mutation of the SIM of FANCI disrupts UAF1/FANCI binding and inhibits FANCD2 deubiquitination. The SLD2 sequence of UAF1 also binds to a SIM on hELG1, and targets the USP1/UAF1 complex to its PCNA-Ub substrate. We proposed the regulated targeting of USP1/UAF1 to its DNA repair substrates, FANCD2-Ub and PCNA-Ub, by SLD-SIM interactions coordinates HR and TLS. Originating from USP1/UAF1 complex, we worked out a general mechanism of DUB regulation by WD40 proteins, which involved in two more DUBs, USP12 and USP46 (discussed in Chapter 4 and Appendix A). Lastly in Chapter 5, through bioinformatic analysis we identified a series of novel proteins containing ubiquitin-binding zinc fingers (UBZ). We then focused on SNM1A and FAAP20/C1orf86, and characterized their function in DNA crosslink repair.

The effect of a single genetic mutation can vary greatly between different types of cells. The mutated gene may not be expressed in one tissue but may cause a devastating loss of function in another. To learn about disease mechanisms and generate novel therapies, genetic disorders must be studied in the types of cells where the mutations are most deleterious. Recently, scientists have begun manipulating cellular identity to create the cell types most affected by various genetic diseases. This dissertation describes the experience of generating reprogramming models for three genetic disorders: Ring 14 syndrome, Pearson syndrome, and Fanconi anemia.

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.

Tese (doutorado) - Pontifícia Universidade Católica do Paraná, Curitiba, 2010 / Bibliografia: f. 41-44 / Anemia de Fanconi (AF) é uma doença genética autossômica recessiva, com falência da medula óssea, malformações congênitas ósseas: anomalias de crânio, face e membros superiores atraso do crescimento e associação a neoplasias. Este estudo avaliou o padrão / Fanconi Anemia (FA) is an autosomal recessive genetic disease, with bone marrow failure, congenital osseous anomalies such as skull, face and upper limb anomalies, growth delay and it presents association with neoplasia. This study evaluated craniofacial

Dissertação (mestrado) - Pontifícia Universidade Católica do Paraná, Curitiba, 2009 / Bibliografia: f. 20-22 / OBJETIVOS: Avaliar e correlacionar fatores de risco à doença cárie, como dieta, higienização bucal e microbiota bucal potencialmente cariogênica (Streptococcus mutans) com experiência de doença cárie por meio de levantamento de índice CPO-D/ ceo-d em paci / OBJECTIVES: This study was aimed to evaluate and to correlate risk factors for the caries disease with caries experiences by DMFT / dmft index in patients presenting with Fanconi´s anemia (FA). MATERIAL AND METHOD: 31 FA patients aged between 4 to 20 were