APLF (Aprataxin and Polynucleotide kinase-Like Factor), a novel protein with a forkhead-associated (FHA) domain and two poly(ADP-ribose)-binding zinc fingers (PBZ), interacts with core non-homologous end-joining (NHEJ) repair factors, Ku and XRCC4-DNA ligase IV, and facilitates NHEJ. However, how APLF functions in NHEJ is undefined. This thesis demonstrates that the Ku-binding domain on APLF is mapped to amino acid residues 180-200, where conserved amino acid residue W189 strongly contributes to the APLF-Ku interaction. Remarkably, the APLF-Ku interaction is involved in the nuclear localization of APLF. Furthermore, we demonstrate that the N-terminal region (amino acids 1-200), containing the XRCC4-Ligase IV and Ku binding domains, is required for APLF- dependent NHEJ. Collectively, these findings suggest that Ku contributes to APLF nuclear localization, and that once APLF is retained in the nucleus, the N-terminal portion of APLF, which facilitates interactions with the core NHEJ proteins Ku and XRCC4-DNA ligase IV, is required for efficient NHEJ.

APLF (Aprataxin and Polynucleotide kinase-Like Factor), a novel protein with a forkhead-associated (FHA) domain and two poly(ADP-ribose)-binding zinc fingers (PBZ), interacts with core non-homologous end-joining (NHEJ) repair factors, Ku and XRCC4-DNA ligase IV, and facilitates NHEJ. However, how APLF functions in NHEJ is undefined. This thesis demonstrates that the Ku-binding domain on APLF is mapped to amino acid residues 180-200, where conserved amino acid residue W189 strongly contributes to the APLF-Ku interaction. Remarkably, the APLF-Ku interaction is involved in the nuclear localization of APLF. Furthermore, we demonstrate that the N-terminal region (amino acids 1-200), containing the XRCC4-Ligase IV and Ku binding domains, is required for APLF- dependent NHEJ. Collectively, these findings suggest that Ku contributes to APLF nuclear localization, and that once APLF is retained in the nucleus, the N-terminal portion of APLF, which facilitates interactions with the core NHEJ proteins Ku and XRCC4-DNA ligase IV, is required for efficient NHEJ.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Less than half of patients with epithelial ovarian cancer (EOC) survive five years following diagnosis, underscoring the imperative need for improved treatment. Many patients, including those with advanced disease, initially respond to platinum agents, which constitute the backbone of therapy. However, tumors ultimately become resistant, rendering further treatment ineffective. Additionally, the poor tolerability of these agents warrants the exploration of more targeted treatments – one such strategy is exploiting synthetic lethal genetic relationships. Recent genomic sequencing efforts have revealed that as many of half of EOCs have homologous recombination (HR) alterations. HR is a critical pathway for the repair of platinum-induced ICLs, thus compromised HR is hypothesized to explain the initial response to chemotherapy in many patients. Accordingly, women whose tumors harbor mutations in the critical HR genes, BRCA1 or BRCA2 (BRCA1/2), demonstrate improved prognosis. BRCA1/2 mutations also confer exquisite sensitivity to inhibitors of the enzyme, poly(ADP-ribose) polymerase 1 (PARPis), hence loss of BRCA1/2 and PARP1 is synthetic lethal. A number of models have been proposed to explain this synthetic lethality, yet a consensus model that accounts for the diverse cellular roles of BRCA1/2 and PARP1 has yet to be established. Delineating the precise molecular underpinnings of PARPi action in BRCA1/2-deficient cells will aid clinicians in identifying the appropriate population of women with EOC likely to benefit from PARPi treatment and provide insight into resistance mechanisms that arise in these patients. Combining this approach with retrospective analysis of PARPi clinical trials will best define the proper indication for PARPi in EOC and other human cancers.

L’ADN est constamment exposé à des insultes génotoxiques endogènes et exogènes. Plusieurs mécanismes de réparations de l’ADN sont mis en œuvre pour préserver la stabilité du génome et de l’épigénome. La recombinaison homologue (RH) joue un rôle central dans la réparation des cassures double brin de l’ADN (DSBs) et le redémarrage des fourches de réplication en réponse à un stress réplicatif. Ces deux processus sont tous deux couplés à l’assemblage de la chromatine. Le facteur d’assemblage de la chromatine 1 (CAF-1) est un chaperon d’histone conservé au cours de l’évolution qui fonctionne dans le processus d’assemblage des nucléosomes couplé à la réparation de l’ADN et à la réplication, en déposant sur l’ADN les tétramères d’histones (H3-H4)2 nouvellement synthétisés. Chez la levure Schizosaccharomyces pombe, le complexe CAF-1 est constitué de trois sous-unités, Pcf1, Pcf2 et Pcf3. Il a été montré que CAF-1 agit dans l’étape de synthèse de l’ADN durant le processus de réplication dépendante de la recombinaison (RDR) et protège le désassemblage des D-loop par l’hélicase Rqh1, membre de la famille des hélicases RecQ. Dans cette étude, nous avons adressé le rôle de CAF-1 pendant la réparation de l’ADN par recombinaison homologue chez la levure Schizosaccharomyces pombe. Par l’utilisation d’approches in vivo et in vitro, nous avons validé des interactions protéines-protéines au sein d’un complexe contenant Rqh1, CAF-1, PCNA, et l’Histone H3. Nous avons montré que Rqh1 interagit avec Pcf1 et avec Pcf2 indépendamment l’un de l’autre, et que l’interaction Rqh1-Pcf1 est stimulée par des dommages à l’ADN. Nous avons mis en place une méthode d’analyse de liaison à la chromatine pour suivre l’association de CAF-1 à la chromatine en réponse aux dommages à l’ADN. Nous avons observé qu’un stress réplicatif, mais pas l’induction de cassures double brin de l’ADN, favorise l’association de CAF-1 à la chromatine. Nous avons identifié plusieurs facteurs de la RH nécessaire pour l’association de CAF-1 à la chromatine en réponse à un stress réplicatif. De plus, nous avons mis en évidence des interactions physiques entre Pcf1 et des facteurs de la recombinaison homologue, parmi lesquels RPA et Rad51. Nos données suggèrent que CAF-1 pourrait s’associer aux sites de synthèse d’ADN dépendent de la recombinaison via son interaction avec des facteurs de la RH. L’ensemble des données de cette étude contribuent à renforcer le role de CAF-1 couplé à réparation de l’ADN, et révèlent une interconnexion entre les facteurs de la RH et l’assemblage de la chromatine. / DNA is constantly exposed to both endogenous and exogenous genotoxic insults. Multiple DNA repair mechanisms are exploited to guard the genome and epigenome stability. Homologous recombination (HR) plays a major role in repairing DNA double strand breaks (DSBs) and restarting stalled replication forks under replicative stress. These two processes are both coupled to chromatin assembly. Chromatin assembly factor 1 (CAF-1) is a highly conserved histone chaperone known to function in a network of nucleosome assembly coupled to DNA repair and replication, by depositing newly synthesized histone (H3-H4)2 tetramers onto the DNA. The fission yeast CAF-1 complex consists of three subunits Pcf1, Pcf2 and Pcf3. CAF-1 has been previously reported to act at the DNA synthesis step during the process of recombination-dependent replication (RDR) and protects the D-loop from disassembly by the RecQ helicase family member, Rqh1. In this study, we addressed the role of CAF-1 during homologous-recombination-mediated DNA repair in fission yeast.Using in vivo and in vitro approaches, we validated interactions within a complex containing Rqh1, CAF-1, PCNA, and Histone H3. We showed that Rqh1 interacts with both Pcf1 and Pcf2 independently of each other, and the Pcf1-Rqh1 interaction is stimulated by DNA damage. We developed an in vivo chromatin binding assay to monitor the association of CAF-1 to the chromatin upon DNA damage. We observed that replication stress but not double strand break favors CAF-1 association to the chromatin. We identified that several HR factors are required for CAF-1 association to the chromatin upon replication stress. In support of this, we have identified physical interactions between Pcf1 and HR factors, including RPA and Rad51. Our data suggest that CAF-1 would associate with the site of recombination-dependent DNA synthesis through physical interactions with HR factors. Put together, this work contributes to strengthening the role of CAF-1 coupled to DNA repair, and reveals the crosstalk between HR factors and chromatin assembly.

No description available.

The aims of the present investigation are fourfold: (i) to review the range of non-invasive methods that may be used to diagnose acute cardiac allograft rejection; (ii) to review the use of the bioptome in sampling the donor heart endomyocardium; (iii) to review the light microscopic and histological grading of acute cardiac rejection; (iv) to characterise the mononuclear populations in endomyocardial biopsy samples and correlate the findings with the light microscopic appearances of the same biopsy specimens.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20668号 / 医博第4278号 / 新制||医||1024(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 武田 俊一, 教授 岩井 一宏, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand breaks (DSB), particularly those induced by ionizing radiation (IR) are complex lesions and if not repaired, these breaks can lead to genomic instability, chromosomal abnormalities and cell death. IR-induced DSB often have DNA termini modifications including thymine glycols, ring fragmentation, 3' phosphoglycolates, 5' hydroxyl groups and abasic sites. Non-homologous end joining (NHEJ) is a major pathway responsible for the repair of these complex breaks. Proteins involved in NHEJ include the Ku 70/80 heterodimer, DNA-PKcs, processing proteins including Artemis and DNA polymerases µ and λ, XRCC4, DNA ligase IV and XLF. The precise molecular mechanism of DNA-PK activation and Artemis processing at the site of a DNA DSB has yet to be elucidated. We have investigated the effect of DNA sequence and structure on DNA-PK activation and results suggest a model where the 3' strand of a DNA terminus is responsible for annealing and the 5' strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ. Artemis, a nuclease implicated in processing of DNA termini at a DSB during NHEJ, has been demonstrated to have both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity. Evidence suggests that either the enzyme contains two different active sites for each of these distinct processing activities, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. These results reveal novel mechanisms of two critical NHEJ proteins, and further enhance our understanding of DNA-PK and Artemis activity and their role in NHEJ.