La voie de réparation NHEJ (Non-Homologous End-Joining) est une voie majeure de réparation des cassures double-brin chez l’homme. Les protéines de cette voie interagissent et forment des complexes dynamiques dont les mécanismes moléculaires sont encore largement méconnus. Nous avons dans un premier temps mis au point des protocoles de production à l’échelle de plusieurs milligrammes des protéines cœur de la voie NHEJ en cellules d’insecte à l’aide du système MultiBac. Nous avons ainsi purifié les complexes Ku70/Ku80 et Ligase4/XRCC4 et les protéines Cernunnos et Artemis à homogénéité. Des essais de cristallisation, des études par SAXS et des analyses par microscopie électronique ont été réalisés sur différents complexes formés par ces protéines cœur du NHEJ. Nous avons également caractérisé par chromatographie d’exclusion de taille et calorimétrie, les interactions effectuées entre les protéines de la voie NHEJ. L’ensemble de ces travaux a permis d’établir des bases biochimiques solides en vue des études structurales et fonctionnelles de la voie NHEJ chez l’homme. / Human DNA repair pathway NHEJ (Non-Homologous End-Joining) is a major pathway of double-strand breaks repair. The proteins involved in this pathway interact and form dynamic complexes whose molecular mechanisms are largely unknown. Firstly, we established protocols to be able to purify milligrams of those NHEJ pathway core proteins using MultiBac insect cells system. We then purified Ku70/Ku80 and Ligase4/XRCC4 complexes, Artemis and Cernunnos to homogeneity. Crystallogenesis assays, SAXS experiments and Transmission Electronic Microscopy experiments have been performed on several complexes formed by these core NHEJ proteins. We also characterized the interactions between these proteins by Size Exclusion Chromatography and Isothermal Calorimetry. These experiments have led to biochemical results sufficient to establish a solid basis to initiate the structural and functional study of the Human NHEJ Pathway.

Free radical-mediated DNA double strand breaks (DSBs) are induced either directly by ionizing radiation or by certain chemicals like bleomycin. These breaks are terminated by 3'-PG (PO4CH2COOˉ) or 3'-phosphate groups formed as a result of fragmentation of deoxyribose. To study the nature of repair of these 3'-blocked breaks, we constructed substrates mimicking free-radical induced DSBs. Human and yeast tyrosyl DNA-phosphodiesterase (Tdpl) efficiently processed substrates with 3'-PGs, in either the presence or absence of magnesium, to give a 3'-phosphate. Gel filtration chromatography and western blotting codmed that the putative enzyme in human extracts that efficiently processed PG was indeed tyrosyl DNA-phosphodiesterase. When recombinant hTdpl was purified using HiTrap nickel chelating columns and its PG processing activity compared to that of partially purified native enzyme (from lymphoblastoid whole-cell extracts using Sephacryl S-300 gel filtration columns), we found that the recombinant enzyme had lesser 3'-PG removal activity than the partially purified native enzyme. On cloning recombinant FLAG-tagged hTdpl into human expression vectors, we observed that the FLAG epitope tag did not show any evidence of affecting the specificity of the enzyme. Due to the many differences between bacterial and human cells, we cloned recombinant FLAG-tagged hTdpl into U-87 cells (adenovirus infected glioma cell) and this recombinant enzyme showed the same specificity toward PG substrates as when prepared from bacteria. End-processing assays using the NHEJ proteins- Ku, DNA-PK and XRCC4/Ligase IV-alone or in combination showed an inhibition of hTdpl activity on 3'- overhangs. In nuclear extracts, hTdp1 association with XRCC1, a single-strand repair protein, showed to increase the PG-processing activity of Tdpl up to 4 times. Whole-cell extracts containing mutant Tdpl derived from patients suffering from spinocerebellar axonal neuropathy (SCAN1) were found to be deficient in PG-processing. Addition of JRLl whole-cell extract (SCAN1 extract containing mutant Tdpl) to purified FLAG-tagged hTdpl showed to decrease the phosphotyrosyl processing and increase the PG-processing of FLAG-tagged hTdpl suggesting that there must be other factors in the extract that affect the enzyme activity. Experiments carried out to check for the presence of Tdpl in mitochondrial extracts obtained from GM1310 normal human fibroblasts as well as in SCANl (JRL) mitochondrial extracts, showed that mitochondrial extracts contained Tdpl at a concentration comparable to whole-cell extracts. Our results also showed that mitochondrial extracts from the SCANl cell-line, JRL3 (containing mutant Tdpl), lacked detectable Tdpl activity suggesting that all PG-processing activity in mitochondria may be attributable to Tdpl.

Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase λ (pol λ), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol λ can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol λ, we also show that the unique proline-rich region in pol λ plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol λ interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol λ. Taken together, these data strongly support that pol λ functions in DNA polymerization events during NHEJ.

Fundamental to mammalian brain development is the integration of cell intrinsic and extrinsic signals that direct the proliferation and differentiation of neural stem cells. Precise expression of transcription factors together with other intracellular components instruct progenitor cell fate, whereas interaction with extracellular signaling factors refines this process. We have elucidated the composition of the cerebrospinal fluid that is the source of multiple extrinsic cues during brain development. The choroid plexus, a highly vascularized tissue located in each ventricle of the brain, actively secretes cerebrospinal fluid. By RNA sequencing, we obtained transcriptome data on the choroid plexi from lateral and fourth ventricles of the mouse brain and discovered that they include transcripts unique to each tissue. Transcription factor expression in the macaque and human choroid plexi suggests that positional identities of these tissues are conserved in the primate brain. Based on transcriptional results, we defined the choroid plexus secretome, a prediction of secreted factors from the choroid plexus. By quantitative mass spectrometry, we detected proteins secreted by each choroid plexus, and comparison of these proteomic results with transcriptional profiling suggests that choroid plexus transcriptomes contribute to availability of regionalized cerebrospinal fluid factors during development. In the second part of my dissertation research, I studied the role of DNA repair mechanisms in regulating neural stem cells. These studies focused on DNA LigaseIV, an essential component of DNA double-stranded break repair, during cerebral cortical development. Deficiency of LigaseIV activity caused by a missense mutation leads to LigaseIV syndrome, in which a key clinical feature is microcephaly. Using the Lig4 R278H mouse mutant, we found increased cell death in the developing cortex, contributing to reduced cortical thickness and cellularity in the anterior cerebral cortex. These results indicate that DNA LigaseIV is essential for proper cortical development. Together, these findings illustrate the complexity of regulatory mechanisms that guide brain development, requiring the integration of mechanisms from within and outside the cell. We have investigated two such mechanisms, extrinsic cues from regionalized cerebrospinal fluid and DNA LigaseIV. These results should provide greater insight into mechanisms of normal brain development and neuropathological states. / 2017-11-02T00:00:00Z

<p> Non-homologous end joining (NHEJ) is the predominant mode of DNA double strand break (DSB) repair pathway in mammalian cells. At the heart of this repair pathway is Xrcc4-DNA ligase IV complex, which mediates ligation of the broken DNA strands. The C-terminal tandem BRCT repeats of human DNA ligase IV spanning residues 654-911 in complex with the functional fragment of Xrcc4 comprised of residues 1-203 were crystallized by the hanging drop vapour diffusion method at 20°C. Generation of single, well-packed, diffraction quality crystals suitable for structure determination involved usage of an Xrcc4 point mutant (A60E). Arriving at the crystallization condition included optimization of pH, variation of the precipitant concentration, investigation of the effects of small molecules, and alteration of the amount of crystal seed used as initial nuclei. A Crystal of selenomethionine-derived protein complex was grown using the above optimization steps and diffracted to 2.4 A resolution. Data processing revealed that the crystal belonged to space group P1 with unit cell dimensions a= 67.33 b = 86.00 c = 111.52; a= 67.37 ~ = 83.00 y = 74.56. The crystal structure of Xrcc4-DNA ligase IV complex was solved by single-wavelength anomalous diffraction using data collected at a wavelength of 0.9785A corresponding to peak energy. </p> <p> The structure maintains a 2:1 stoichiometry of Xrcc4 to the C-terminal domain of DNA ligase IV. The structure of the complex not only confirms the overall novel mode of interaction first observed in the 3.9 A structure of the yeast ortholog liflp-lig4p complex, but it also discloses additional key features such as the DNA binding surface of the complex and the striking conformational changes occurring within Xrcc4 upon interaction with DNA ligase IV. Together, the structural information procured forms an important basis for a better understanding of the mechanism involved in the NHEJ repair pathway. </p> / Thesis / Master of Science (MSc)

Les défauts dans la réparation des cassures double-brin de l'ADN (DSBs) peuvent avoir d'importantes conséquences pouvant entrainer une instabilité génomique et conduire à la mort cellulaire ou au développement de cancers. Dans la plupart des cellules mammifères, le mécanisme de Jonction des Extrémités Non Homologues (NHEJ) est le principal mécanisme de réparation des DSBs. L'ADN Ligase IV (LigIV) est une protéine unique dans sa capacité à promouvoir la NHEJ classique. Elle s'associe avec deux autres protéines structuralement similaires, XRCC4 et XLF (ou Cernunnos). LigIV interagit directement avec XRCC4 pour former un complexe stable, tandis que l'interaction entre XLF et ce complexe est médiée par XRCC4. XLF stimule fortement l'activité de ligation du complexe LigIV/XRCC4 par un mécanisme encore indéterminé. Récemment, un rôle structurel non catalytique a été attribué à LigIV (Cottarel et al., 2013). Dans le travail de thèse présenté ici, nous avons reconstitué l'étape de ligation de la NHEJ en utilisant des protéines recombinantes produites dans des bactéries afin d’une part, d'explorer les bases moléculaires du rôle structural de LigIV, d’autre part de comprendre le mécanisme par lequel XLF stimule le complexe de ligation, et enfin de mieux comprendre comment ces trois protéines coopèrent au cours de la NHEJ. Nos analyses biochimiques suggèrent que XLF via son interaction avec XRCC4 lié à LigIV, pourrait induire un changement conformationnel dans la LigIV. Ce réarrangement de la ligase exposerait son interface de liaison à l'ADN ce qui lui permettrait alors de ponter deux molécules indépendantes d'ADN, une capacité indépendante de l'activité catalytique de LigIV. / Failure to repair DNA double-strand breaks (DSBs) may have deleterious consequences inducing genomic instability and even cell death. In most mammalian cells, Non-Homologous End Joining (NHEJ) is a prominent DSB repair pathway. DNA ligase IV (LigIV) is unique in its ability to promote classical NHEJ. It associates with two structurally related proteins called XRCC4 and XLF (aka Cernunnos). LigIV directly interacts with XRCC4 forming a stable complex while the XLF interaction with this complex is mediated by XRCC4. XLF strongly stimulates the ligation activity of the LigIV/XRCC4 complex by an unknown mechanism. Recently, a structural noncatalytic role of LigIV has been uncovered (Cottarel et al., 2013). Here, we have reconstituted the end joining ligation step using recombinant proteins produced in bacteria to explore not only the molecular basis for the structural role of LigIV, but also to understand the mechanism by which XLF stimulates the ligation complex, and how these three proteins work together during NHEJ. Our biochemical analysis suggests that XLF, through interactions with LigIV/XRCC4 complex, could induce a conformational change in LigIV. Rearrangement of the LigIV would expose its DNA binding interface that is able to bridge two independent DNA molecules. This bridging ability is fully independent of LigIV’s catalytic activity. We have mutated this interface in order to attempt to disrupt the newly identified DNA bridging ability. In vitro analysis of this LigIV mutant will be presented as well as a preliminary in vivo analysis.

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