Benzene is an ubiquitous pollutant and industrial solvent that has been identified as a human leukemogen. Early exposure to environmental carcinogens such as benzene has been postulated to play a role in the etiology of childhood leukemia, however the association remains controversial. Genotoxic agents such as benzene can cause an increase in the frequency of DNA double-strand breaks, which may remain unrepaired or result in the initiation of DNA recombinational repair mechanisms. The first objective was to investigate the induction of DNA double-strand breaks following in utero treatment to 200 mg/kg and 400 mg/kg benzene i.p. using the phosphorylated histone γ-H2A.X as a marker. Using immunoblotting, treatment with benzene did not increase the formation of γ-H2A.X in bone marrow cells of adult C57Bl/6N male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells following in utero exposure to 200 mg/kg or 400 mg/kg benzene throughout gestational days 7 to 15. Secondly, the study investigated the induction of micronuclei following in utero exposure to benzene. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells. In utero exposure to 400 mg/kg benzene throughout gestational days 7 to 15 also caused a statistically significant increase in the percentage of micronucleated cells in maternal bone marrow and post-natal bone marrow cells. Fetal liver cells also demonstrated a statistically significant increase in the percentage of micronucleated cells following 200 mg/kg and 400 mg/kg benzene. The third objective was to investigate the initiation of DNA recombination following in utero exposure to benzene using the pKZ1 mutagenesis mouse model as a surrogate marker for non-homologous end joining activity. Adult pKZ1 mouse tissue yielded no recombination events; however, post-natal bone marrow cells did contain detectable recombination frequencies. iii In utero benzene exposure did cause an increasing trend in recombination events, and upon analysis of only the samples containing detectable levels of recombination, in utero exposure to 400 mg/kg of benzene caused a statistically significant increase in recombination frequency within this group. These results demonstrate that benzene does not increase the formation of γ-H2A.X after acute and in utero exposure, however, the induction of micronuclei following acute and in utero benzene exposure confirmed that benzene is a genotoxic agent causing chromosomal breaks. In utero benzene exposure increased the frequency of DNA recombination in bone marrow from post-natal day 9 pups exhibiting detectable levels of recombination. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood leukemias. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-08-22 11:07:49.162

Au cours de la réplication, la réparation des cassures double brin (CDB) par recombinaison homologue (RH), basée sur la synthèse d’ADN à partir de la chromatide sœur, permet le maintien de la stabilité du génome. La religature d’extrémités (EJ) éloignées de CDB peut quant à elle générer des réarrangements menaçant son intégrité. Nous avons étudié le mécanisme de réparation par EJ en fonction de la distance séparant deux cassures double brin. En utilisant des substrats intra-chromosomiques permettant la mesure de l’efficacité et de la fidélité du EJ après ligature d’extrémités éloignées ou proximales, nous avons mis en évidence l’implication du complexe de cohésion dans l’inhibition du EJ d’extrémités distales. Le complexe de cohésion joue donc un rôle central dans l’interface réplication/réparation ; la cohésion des chromatides sœurs favorise la réparation par RH et permet l’inhibition spécifique du EJ d’extrémités éloignées, probablement en limitant la mobilité de la chromatine endommagée et la formation d’une synapse propice au rapprochement des extrémités. La religature d’extrémités éloignées est également nécessaire aux mécanismes de diversification des gènes des immunoglobulines tels que la recombinaison V(D)J et la commutation de classe. L’étude de souris Rad21+/- a également démontré une implication du complexe de cohésion dans ces mécanismes essentiels à la diversité de l’information génétique. Le complexe de cohésion étant impliqué dans ces mécanismes et dans l’inhibition des réarrangements complexes tels que les translocations et insertions il est un acteur essentiel de la diversité et de la stabilité génomique. / DNA double-strand breaks (DSBs) repair is essential for genome stability/diversity, but can also generate genome rearrangements. Although non-homologous end-joining (NHEJ) is required for genome stability maintenance, the joining of distant double strand ends (DSE) should inexorably lead to genetic rearrangements. We analyzed the efficiency and accurency of close or distal EJ repair. Our data show that global end-joining is more efficient on close ends (34bp) compared to distal ends (3200bp) and that C-NHEJ is favored on close ends, resulting in more accurate outcome, compared to distal ends where more mutagenic A-EJ events takes place. In addition, the joining of distal ends favors the insertion/capture of DNA sequences. These data show only few kb distances between two DSEs are sufficient to jeopardize DSB repair efficiency and accuracy, leading to complex scars at the re-sealed junctions, and cell response is sufficiently sensitive to differently process such distal ends. We next addressed the question of the mechanisms preventing the joining of distant DSE. We show that depletion of the cohesin complex proteins specifically stimulates the end-joining of I-SceI-induced DSBs distant of 3200bp, while the joining of close DSEs (34bp) remained unaffected. Consistently, exome sequencing and cytogenetic analysis revealed that RAD21 ablation generates large chromosome rearrangements and a strong induction of replication stress-induced chromosome fusions. These data reveal a role for the cohesin complex in the protection against profound genome rearrangements arising through ligation of distant DSEs.

Eukaryotic cells repair DNA double-strand breaks (DSBs) through both non-homologous and homologous recombination pathways. The initiation of homologous recombination requires the generation of 3' overhangs, which are essential for the formation of Rad51 protein-DNA filaments that catalyze subsequent steps of strand invasion. Experiments in budding yeast show that resection of the 5' strand at a DSB is delayed in strains lacking any components of the Mre11/Rad50/Xrs2 (MRX) complex¹ . In meiosis, a specific class of hypomorphic mutants of mre11 and rad50 (Rad50S) are completely deficient in 5' resection and leave Spo11 covalently attached to the 5' strands of DNA breaks². Similar to mre11S and rad50S mutants, sae2 deletion strains fail to resect 5' strands at meiotic DSBs and accumulate covalent Spo11 adducts³;⁴. In addition, Sae2 and MRX were also found to function cooperatively to process hairpin-capped DNA ends in vivo in yeast. sae2 and mrx null strains show a severe defect in processing these structures and accumulate hairpin-capped DNA ends⁵;⁶. The Longhese laboratory has also shown that Sae2 deletion strains show a delay in 5' strand resection, similar to rad50S strains⁷. Recently, Bettina Lengsfeld in our laboratory demonstrated that Sae2 itself possesses nuclease activity and that MRX and Sae2 act cooperatively to cleave single-stranded DNA adjacent to DNA hairpin structures⁸. In vitro characterization of Sae2 showed that the central and N-terminal domains are required for MRX-independent nuclease activity and that the C-terminus is required for cooperative activities with MRX. Sae2 also acts independently of MRX as a 5' flap endonuclease on branched structures in vitro. Our studies investigate whether MRX, Sae2, and Exo1 function cooperatively in DNA resection using recombinant, purified proteins in vitro. We developed assays utilizing strand-specific Southern blot analysis to visualize DNA end processing of model DNA substrates using recombinant proteins in vitro. Our results demonstrate that MRX and Sae2 cooperatively resect the 5' end of a DNA duplex together with the Exo1 enzyme, supporting a role for these factors in the early stages of homologous recombination and repair. / text

DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. To protect genomic integrity and ensure cellular homeostasis, cells initiate a complex signaling-based response that activates cell cycle checkpoints, coordinates DNA repair, regulates gene expression and, if necessary, induces apoptosis. The spatio-temporal control of this signaling pathway relies on a large number of post-translational modifications, including phosphorylation and regulatory ubiquitylation. In this thesis, I describe the discovery and characterization of the E3 ubiquitin ligase RNF168, which cooperates with the upstream E3 ubiquitin ligase RNF8 to form a cascade of regulatory ubiquitylation at damaged chromatin. One of the main functions of RNF8/RNF168-dependent chromatin ubiquitylation is to generate a molecular landing platform for the ubiquitin-dependent accumulation of checkpoint and DNA repair proteins such as 53BP1, the breast-cancer associated protein BRCA1 and the RNF168-paralog RNF169. I present evidence that the hierarchical recruitment of these proteins to DSB sites is, in large part, organized through the use of tandem protein interaction modules. These modules are composed of a ubiquitin-binding domain and an adjacent targeting motif called LRM, which specifies the recognition of RNF8- and RNF168-ubiquitylation substrates at damaged chromatin. I conclude that the LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the chromatin-based response to DSBs. Collectively, my results indicate that RNF168-mediated chromatin ubiquitylation is critical for the physiological response to DSBs in human cells. The importance of the ubiquitin-based response to DSBs is underscored by the finding that RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder, is caused by mutations in the RNF168 gene.

DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. To protect genomic integrity and ensure cellular homeostasis, cells initiate a complex signaling-based response that activates cell cycle checkpoints, coordinates DNA repair, regulates gene expression and, if necessary, induces apoptosis. The spatio-temporal control of this signaling pathway relies on a large number of post-translational modifications, including phosphorylation and regulatory ubiquitylation. In this thesis, I describe the discovery and characterization of the E3 ubiquitin ligase RNF168, which cooperates with the upstream E3 ubiquitin ligase RNF8 to form a cascade of regulatory ubiquitylation at damaged chromatin. One of the main functions of RNF8/RNF168-dependent chromatin ubiquitylation is to generate a molecular landing platform for the ubiquitin-dependent accumulation of checkpoint and DNA repair proteins such as 53BP1, the breast-cancer associated protein BRCA1 and the RNF168-paralog RNF169. I present evidence that the hierarchical recruitment of these proteins to DSB sites is, in large part, organized through the use of tandem protein interaction modules. These modules are composed of a ubiquitin-binding domain and an adjacent targeting motif called LRM, which specifies the recognition of RNF8- and RNF168-ubiquitylation substrates at damaged chromatin. I conclude that the LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the chromatin-based response to DSBs. Collectively, my results indicate that RNF168-mediated chromatin ubiquitylation is critical for the physiological response to DSBs in human cells. The importance of the ubiquitin-based response to DSBs is underscored by the finding that RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder, is caused by mutations in the RNF168 gene.

During my PhD, I have investigated aspects of the DNA damage response (DDR) in the context of three different cellular scenarios: DNA damage signalling in response to double-strand breaks during mitosis, coordination of DNA replication with DNA damage responses by regulation of the GINS complex, and checkpoint activation by the prototypical checkpoint protein Rad9. Here, I show that mitotic cells treated with DNA break-inducing agents activate a 'primary' DDR, including ATM and DNA-PK-dependent H2AX phosphorylation and recruitment of MDC1 and the MRN complex to damage sites. However, downstream DDR events and induction of a DNA damage checkpoint are inhibited in mitosis, with full DDR activation only ensuing when damaged mitotic cells enter G1. In addition, I provide evidence that induction of a primary DDR in mitosis is biologically important for cell viability. The GINS complex is an evolutionarily conserved component of the DNA replication machinery and may represent an ideal candidate for transferring the DNA damage signal to the replication apparatus. Here, I show the identification of a consensus 'SQ' PIKK phosphorylation motif at the carboxyl end of the GINS complex subunit, Psf1. In Saccharomyces cerevisiae, switching the conserved serine to a glutamic acid is lethal, indicating that the site is crucial for the protein's function. Moreover, in human cells, I identified UV-DDB, a heterodimeric complex involved in NER repair, as a binding partner that specifically interacts with the Psf1 C-terminus in vitro. Finally, I discuss my findings in characterizing functional interactions between Rad9 and Chk1 in S. cerevisiae. I show that specific consensus CDK sites within Rad9 N-terminus are essential to enable Chk1 phosphorylation and activation, and that MCPH1, a human homologue of Rad9, may share a conserved function in binding and activating Chk1, underscoring the evolutionarily conservation of checkpoint activation mechanisms.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22730号 / 医博第4648号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 遊佐 宏介, 教授 溝脇 尚志, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23090号 / 医博第4717号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 小川 修, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23114号 / 医科博第125号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 篠原 隆司, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cellular accumulation of DNA damage has been widely implicated in cellular senescence, aging, and premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is linked to accumulation of DNA double-strand breaks (DSBs), which results in genome instability. However, how DSBs accumulate in cells despite the presence of intact DNA repair proteins remains unknown. Here we report that the recruitment of DSB repair factors Rad50 and Rad51 to the DSB sites, as marked by γ-H2AX, was impaired in human HGPS and Zmpste24-deficient cells. Consistently, the progeria-associated DSBs appeared to be unrepairable although DSBs induced by camptothecin were efficiently removed in the progeroid cells. We also found that these progeroid cells exhibited nuclear foci of xeroderma pigmentosum group A (XPA), a unique nucleotide excision repair protein. Strikingly, these XPA foci colocalized with the DSB sites in the progeroid cells. This XPA-DSB association was further confirmed and found to be mediated by DNA, using a modified chromatin immunoprecipitation assay and coimmunoprecipitation. RNA interference (RNAi) knockdown of XPA in HGPS cells partially restored DSB repair as evidenced by Western blot analysis, immunofluorescence and comet assays. We propose that the uncharacteristic localization of XPA to or near DSBs inhibits DSB repair, thereby contributing to the premature aging phenotypes observed in progeria arising from genetic defects in prelamin A maturation.