京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25025号 / 生博第516号 / 新制||生||69(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 鈴木 淳, 教授 中野 雄司, 教授 野田 岳志 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

<p>If not efficiently repaired, DNA double-stranded breaks can result in cell death. A major contributor to the repair of this DNA damage is the non-homologous end joining pathway (NHEJ) which depends on the proteins: X-ray cross complementing protein 4 (XRCC4) and XLF. These proteins form a complex that can bridge DNA substrates <em>in vitro. </em>Analysis of these proteins has demonstrated that the C-terminal region of XRCC4 is necessary for this bridging function. However, this region is also critical for both tetramerization and DNA binding abilities of XRCC4, making the interpretation of XRCC4's role in the DNA-bridging unclear. Here, we intend to further characterize the tetramerization of XRCC4 and find a functionally independent mutant. Our studies suggest that regions in the N-terminus of XRCC4 may be important for the tetramerization of the protein but not for its DNA binding ability. These mutants were also analyzed by circular dichroism and mobility shift assays to verify for the integrity of their secondary structure composition and show that they are able to interact with its known binding partner, DNA Ligase IV. Additionally, we have shown that the XRCC4:XLF complex as well as XLF alone are able to interact with DNA substrates as short as 36 base pairs. Taking the data together, we expect to be able to construct a structural model for the XRCC4:XLF complex with DNA and obtain a better understanding on the role of XRCC4’s tetramerization in the NHEJ pathway. As deficiency of XRCC4 has been implicated with tumourigenesis and immunodeficiency, understanding its role will be helpful for the development of treatments for such complications.</p> / Master of Science (MSc)

Body mass varies considerably between different mammals and this variation is largely accounted for by a difference in total cell number rather than individual cell size. Insights into mechanisms regulating growth can therefore be gained by understanding what governs total cell number at any one point. In addition, control of cell proliferation and programmed cell death is fundamental to other areas of research such as cancer and stem cell research. Microcephalic Primordial Dwarfism (MPD) is a group of rare Mendelian human disorders in which there is an extreme global failure of growth with affected individuals often only reaching a height of around one metre in adulthood. To date, all identified disease genes follow an autosomal recessive mode of inheritance and encode key regulators of the cell cycle, where mutations impact on overall cell number and result in a substantially reduced body size. MPD therefore provides a valuable model for examining genetic and cellular mechanisms that impact on growth. The overall aims of this thesis were to identify novel disease causing genes, as well as provide further characterisation of known disease causing genes, through the analysis of whole exome sequencing (WES) within a large cohort of MPD patients. Following the design and implementation of an analytical bioinformatics pipeline, deleterious mutations were identified in multiple disease genes including LIG4 and XRCC4. Both genes encode components of the non-homologous end joining machinery, a DNA repair mechanism not previously implicated in MPD. Additionally, the pathogenicity of novel mutations in subunits of a protein complex involved in chromosome segregation was assessed using patient-derived cells. These findings demonstrate WES can be successfully implemented to identify known and novel disease causing genes within a large heterogeneous cohort of patients, expanding the phenotype of known disorders and improving diagnosis as well as providing novel insights into intrinsic cellular mechanisms critical to growth.

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.

Peptides are a promising class of therapeutic candidates due to their high specificity and affinity for cellular protein targets. However, peptides are susceptible to protease degradation and are typically not cell-permeable. In efforts to design more effective peptide drug discovery systems, investigators have discovered that incorporation of non-canonical amino acids (ncAAs) and macrocyclization overcome these limitations, making peptides more drug-like. In this work, we exploit the promiscuity of wild-type aminoacyl-tRNA synthetases (aaRSs) to ‘mischarge’ ncAAs onto tRNA and ribosomally incorporate them into peptides using a cell-free translation system. We have demonstrated the ability to incorporate five ncAAs into a single peptide with near-wild type yield and fidelity. We also demonstrated the in situ incorporation of ncAAs containing azide and alkyne functionalities, enabling the use of CuAAC (click chemistry) to generate triazole-bridged cyclic peptides. When combined with bisalkylation of peptides containing two cysteines via an α,α’-dibromo-m-xylene linker, we created bicyclic peptides which are structurally similar to the highly bioactive knotted peptide natural products. Biological display methods, such as mRNA display, are powerful peptide discovery tools based on their ability to generate libraries of >1014 unique peptides. We combined our ability to incorporate ncAAs with our bicyclization technique adapted for use with mRNA display to create knotted peptide library scaffolds. We performed side-by-side monocyclic and bicyclic in vitro selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nM affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance. We used a new library that enables the generation of a diverse collection of linear, monocyclic and bicyclic scaffolds in one pot, increasing the likelihood of target-ligand conformational alignment. We performed a second selection against streptavidin and revealed a nearly unanimous preference for linear peptides containing an HPQ motif, a known streptavidin-binding sequence. However, when we used these libraries for in vitro selection against a biological target, DNA repair protein XRCC4, we did not observe convergence. In summary, we have developed a novel technique for production of bicyclic peptide libraries. These highly-constrained protease-stable scaffolds can be used as platforms to identify high affinity, drug-like ligands using mRNA display.

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.

<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.

The teratogenic effects of valproic acid (VPA) are well known, though its teratogenic mechanism remains unknown. VPA induces oxidative stress, which may lead to double strand breaks (DSBs) in DNA. Though the cell may repair this damage via homologous recombination (HR) and non-homologous end joining (NHEJ), repair is not always error-free; genomic instability may arise from gene deletions, amplifications, rearrangements, and loss of heterozygosity. Such alterations may underpin VPAʼs teratogenicity. The present study evaluated VPAʼs ability to induce NHEJ and HR and characterized the changes in expression of two proteins key to HR (RAD51) and NHEJ (XRCC4). Using pKZ1 transgenic mice (C57BL/6 genetic background), we sought to measure NHEJ events via X-gal staining. Although consistent staining was observed in adult male brain (positive control), no staining was observed in embryos 12 or 24 hours after in utero exposure to a teratogenic dose of VPA (500 mg/kg, maternal subcutaneous dose) on gestational day 9 (GD9). To determine whether the lack of staining observed in embryos was due to low/absent expression of key DSB-repair proteins, we measured mRNA/protein expression of RAD51 and XRCC4 in C57BL/6, GD9-exposed embryos and maternal brain. One hour after treatment, XRCC4 was increased at the protein level in brain and embryo. RAD51 was not increased in embryos and not detected in adult brain. These data suggest that embryos do possess the protein mediators of NHEJ and HR and that VPA-induced changes in expression of XRCC4 may influence the type of repair pursued, potentially affecting DSB repair fidelity (accuracy). Determination of fidelity of VPA-induced HR was attempted with the Chinese hamster ovary cell line (CHO33) using DNA sequencing; low template concentration and purity precluded successful sequencing of DNA from recombinant colonies and the assessment of fidelity. Overall, these data demonstrate that the lack of X-gal staining observed in pKZ1 embryos is not due to an underexpression of at least one key protein in the NHEJ pathway. Furthermore, a VPA-induced change in the the type of repair pathway pursued by the embryo may have teratological implications. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-10-15 11:06:30.613