In order to preserve its genomic integrity, an organism needs to detect and repair DNA double-strand breaks (DSBs) in a prompt and accurate fashion. This goal is accomplished by enabling an exquisitely sensitive DSB sensing apparatus as well as multiple and often overlapping pathways for repair. All of these processes are carried out on a highly organized and compacted chromatin substrate in the nucleus. An important question is whether chromatin plays an active role in the process and whether it helps in the signaling or repair of this damage. We have used Chromosome Conformation Capture (3C) to show that there are no large scale changes in chromosome structure at a single site-specific DNA double-strand break, although looping interactions between DSBs and donors can be detected. In a surprising result, we found that 3C detected a nucleus-wide decrease in interactions with the DSB. We have used a combination of 3C, fluorescence microscopy and chromatin immunoprecipitation to show that the decrease in interactions is a result of the relocalization of persistent DSB to the nuclear periphery. We also show that this is dependent on the recruitment of telomerase complex to the DSB, which then interacts with its natural partner in the Inner nuclear membrane, Mps3, and relocalizes the DSB to the periphery. Thus, a DSB that cannot be repaired is shunted into a pathway where the cell attempts to survive by putting a de novotelomere on the broken chromosome. Remarkably, this is not an irreversible phenomenon despite the recruitment of telomerase and the relocalization to the periphery. DSBs which are repaired slowly due to the presence of homology on a different chromosome, or merely usage of a kinetically slower form of repair, undergo this pathway switch, but can still recover and repair the DSB if homology is present. We also show that the role of the periphery is to ensure repair through de novotelomere formation or other non-canonical repair pathways. Indeed, loss of peripheral localization results in a dramatic suppression of the genomic instability of the Slx5/8 mutants, which have been implicated in the persistent DSB response at the Nuclear pores. Thus, the nuclear periphery is a special compartment where DSBs go after they cannot be repaired by canonical pathways. Specialized components such as telomerase, silencing proteins and components of the SUMO pathway, all seem to play roles in the healing of these chromosomes. Importantly, the SUN domain homologues of Mps3 have been shown to play roles similar to their yeast homologues in meiotic bouquet formation through their interactions with telomeres. Thus, they may represent a conserved mechanism for chromosome healing and telomere anchoring, despite the fact that mammalian telomeres are rarely found at the nuclear periphery. Such survival mechanisms may be expected to operate in cancer cells which may or may not have upregulated telomerase expression.

During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2010.

Activation-induced cytidine deaminase (AID) is a key protein required for both class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes. AID is induced in B cells during an immune response. Lack of AID or mutant form of AID causes immunodeficiency; e.g., various mutations in the C terminus of AID causes hyper IgM (HIGM2) syndrome in humans. The C terminal 10 amino acids of AID are required for CSR but not for SHM. During both CSR and SHM, AID deaminates dCs within Ig genes, converting them to dUs, which are then either replicated over, creating mutations, or excised by uracil DNA glycosylase (UNG), leading to DNA breaks in Ig switch regions. Also, the mismatch repair (MMR) heterodimer Msh2-Msh6 recognizes U:G mismatches resulting from AID activity and initiates MMR, which leads to increased switch region double strand breaks (DSBs). DSBs are essential intermediates of CSR; lack of UNG or MMR results in a reduction of DSBs and CSR. The DSBs created in the Sμ and one of the downstream S-regions during CSR are recombined by non-homologous end joining (NHEJ) to complete CSR. Available data suggest that AID is required not only for the deamination step of CSR, but also for one or more of the steps of CSR that are downstream of deamination step. This study investigates the role of C terminus of AID in CSR steps downstream of deamination. Using retroviral transduction into mouse splenic B cells, I show that AID binds cooperatively with UNG and Msh2-Msh6 to the Ig Sμ region, and this depends on the AID C terminus. I also show that the function of MMR during CSR depends on the AID C terminus. Surprisingly, the C terminus of AID is not required for Sμ or Sγ3 DSBs, suggesting its role in CSR occurs during repair and/or recombination of DSBs.

La réparation des cassures double brin (CDB) de l'ADN par recombinaison est essentielle au maintien de l'intégrité du génome de tous les être vivants. Ce processus doit cependant être finement régulé puisque la recombinaison peut générer des mutations ou des réarrangements chromosomiques, parfois extrêmement délétères pour la cellule. Les CDB peuvent être réparées par deux mécanismes : la recombinaison non homologue (ou jonction des extrémités d'ADN) ou la recombinaison homologue (impliquant une homologie de séquence entre les molécules recombinantes). Dans les cellules somatiques, les deux voies principales de recombinaison homologue (RH) sont la voie Synthesis Dependent Strand Annealing (SDSA) dépendante de la recombinase RAD51 et la voie Single Strand Annealing (SSA) indépendante de RAD51. Nos résultats ont d'abord mis en évidence un rôle inattendu de XRCC2, RAD51B et RAD51D - trois paralogues de RAD51 - dans la voie SSA. Nous avons confirmé que la fonction de la protéine XRCC2 dans la voie SSA ne dépend pas de RAD51, ce qui démontre que certains paralogues de RAD51 ont acquis des fonctions indépendantes de la recombinase. La différence de sévérité des phénotypes des mutants individuels ainsi que les analyses d'épistasie menées sur le double et le triple mutant suggèrent des fonctions individuelles de ces protéines au cours du SSA. Nous proposons qu'elles facilitent l'étape d'hybridation des deux séquences complémentaires situées de part et d'autre de la cassure, bien que ceci reste à confirmer par des études in vitro. L'étude des fonctions de l'hétérodimère XPF-ERCC1 - un complexe impliqué dans le clivage des extrémités d'ADN non homologues au cours des voies de RH - a révélé un rôle inhibiteur de ce complexe sur la voie SDSA. Cette action est dépendante de son activité endonucléasique et serait liée au clivage des longues extrémités 3' sortantes réalisant l'invasion d'un duplex d'ADN homologue, l'étape initiale de la voie SDSA. Notre étude a de plus confirmé que le rôle du complexe dépend de la longueur des extrémités non homologues chez Arabidopsis, comme chez les mammifères et la levure. Bien que le complexe XPF-ERCC1 soit essentiel au clivage des longues extrémités d'ADN non homologue, il n'est pas requis à l'élimination des courtes extrémités au cours de la RH. / The repair of DNA double-strand breaks (DSB) by recombination is essential for the maintenance of genome integrity of all living organisms. However, recombination must be finely regulated as it can generate mutations or chromosomal rearrangements, sometimes extremely deleterious to the cell. DSB can be repaired by two classes of recombination mechanism: non-homologous recombination (or DNA End Joining) or homologous recombination (implicating DNA sequence homology between the recombining molecules). In somatic cells, the two main pathways of homologous recombination (HR) are RAD51-dependent Synthesis Dependent Strand Annealing (SDSA) and RAD51-independent Single Strand Annealing (SSA). Our results have demonstrated an unexpected role of XRCC2, RAD51B and RAD51D - three RAD51 paralogues – in the SSA pathway. We confirmed that the function of XRCC2 in SSA does not depend upon RAD51, thus demonstrating that some RAD51 paralogues have acquired RAD51 recombinase-independent functions. The different severities of individual mutant phenotypes and epistasis analyses carried out on the double and triple mutants suggest individual functions of these proteins in SSA recombination. We propose that they facilitate hybridization of the two complementary sequences located on both sides of the break, although this remains to be confirmed by in vitro experiments. Study of the roles of XPF-ERCC1 - a complex involved in the cleavage of non-homologous DNA ends during HR - revealed an inhibitory role of this complex on the SDSA pathway. This is dependent on its endonuclease activity and is probably due to the cleavage of long 3' ends performing the homologous DNA duplex invasion, the initial step of the SDSA pathway. Our analyses also confirmed that the role of the complex depends on the length of the nonhomologous ends, as seen in mammals and yeasts. Although XPF-ERCC1 is essential for the cleavage of long nonhomologous DNA ends, it is not required for the elimination of short ends during HR.

Im Rahmen der Studien zur Wirkung von Bleinitrat auf die Embryonalstadien des afrikanischen Welses Clarias gariepinus wurde zunächst der Einfluß der Besamung auf den Härtungsprozess des Chorions untersucht, um die Bedeutung des gehärteten Chorions als Schutzfunktion im Hinblick auf Schadstoffeinwirkung zu klären. Das Studium der Embryonalentwicklung war erforderlich, um das Ausmaß der Änderung der Normalentwicklung unter dem Einfluß von Bleinitrat bewerten zu können. Im Rahmen der toxikologischen Untersuchungen der Wirkung des Bleinitrats auf die Embryonalstadien wurden folgende biologische Marker (Biomarker) betrachtet: Änderungen in der Entwicklung und der Schlüpfrate, morphologische und histologische Änderungen, sowie biochemische Veränderungen (Änderungen von Stoffwechsel-Enzymaktivitäten) und molekulare Veränderungen (Erfassung von DNA-Schädigungen). Die Exposition der besamten Eier mit Bleinitrat führte zu einer Verlängerung der Inkubationszeit und zu starken Mißbildungen. Der Rückgang der Häufigkeiten der Mißbildungen mit der Zeit ließ die Annahme zu, daß die mißgebildeten Embryonen starben. Im Gegensatz zu den morphologischen Mißbildungen wurden histopathologische Effekte nur bei Embryonen gefunden, die den höchsten Dosierungen (300 µg/l und 500 µg/l Bleinitrat) ausgesetzt waren. Nach dem Schlupf war das Muster der Enzymaktivitäten nach Exposition mit Bleinitrat uneinheitlich; die Aktivität von G6PDH nahm zu, die von LDH nahm ab und die von PK zeigte unregelmäßige Fluktuationen. Die Embryonalstadien zeigten signifikante Dosis-abhängige Antworten über die Zeit, da das Ausmaß der DNA-Schädigungen signifikant mit den Bleinitrat Konzentrationen anstieg. Vor dem Schlupf konnten bei den Embryonen nach Bleinitrat Exposition keine Änderungen in den Enzymaktivitäten gefunden werden und nur geringe DNA-Schädigungen, d.h die toxischen Effekte waren sehr gering. Eine Erklärung könnte die schützende Wirkung der Eihülle gegenüber Schadstoffen sein. Die gewählten Biomarker stellen sensitive Detektionsmethoden für Bleinitrat dar. So könnten sie sich als sinnvolle Bioindikatoren für Ägypten erweisen, da dort zunehmend Umweltverschmutzung mit Blei und Bleiakkumulation in Lebensmitteln zu verzeichnen ist. / In order to study the embryo-toxic effects of lead nitrate of the African catfish Clarias gariepinus, we first had to study the effect of fertilization on the hardening process of the chorion to clarify the role of the hardened chorion on the protection of the embryo from the pollutants. Also we had to study the embryonic development of C. gariepinus for providing us with a model for comparison when normal patterns of development are altered due the exposure to lead nitrate. The present toxicological work focuses on lead toxicity in different developmental stages of C. gariepinus considering different biological markers (biomarkers) comprising changes in the development and hatching rate, morphological and histological changes, biochemical changes (alteration of metabolic enzymes activity) and molecular changes (monitoring of DNA damage). Exposure of fertilized eggs to lead nitrate prolonged the incubation period and caused severe morphological malformations. Since the frequencies of the morphological malformations decreased with time, we conclude a lethal impact and selected mortality of abnormal embryos. Unlike the morphological malformation, histopathological changes were only recorded in embryos exposed to the highest dosages (300 µg/l and 500 µg/l lead nitrate). In the post-hatching stages, the patterns of the enzymes activities after lead exposure varied, G6PDH increased, LDH decreased and PK showed fluctuations. Embryonic stages revealed significant dose-related DNA damage response over time, since the degree of DNA damage increased significantly with higher lead concentrations. No specific response in the activities of the selected enzymes and low DNA damage were recorded in the pre-hatching stage after exposure to the lead nitrate doses. This means the lead nitrate had a minute toxic effect on the pre-hatched embryos. We conclude that, low susceptibility in pre-hatching stages is most probably a consequence of the chorion, which seems to protect the embryos from a range of external pollutants. The selected biomarkers were sensitive detection methods for low-level toxicity of lead nitrate. Thus, these are useful tools for biomonitoring, urgently required in Egypt with regard to increasing environmental deposition of lead and bioaccumulation in human food recently observed.