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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.

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.

<p> Repair of double-strand breaks is critical for the preservation of genomic integrity and cellular viability. A predominant pathway implicated in the repair of such lesions is the evolutionarily conserved non-homologous end-joining (NHEJ) pathway. Among the major constituents of this pathway in Saccharomyces cerevisiae is Nej1, for which a clear biochemical function has not been determined. The results presented in this work demonstrate that Nej1 exhibits a DNA binding activity comparable to Lif1, with an apparent dissociation constant of 1.8 μM. Characterization of the DNA binding activity revealed that although short ~20 bp substrates can suffice, binding is enhanced with longer substrates (>300). This DNA binding activity supports the hypothesis that Nej1 plays a direct role in the repair of DNA double-strand breaks. Structure-function studies indicated that the C-terminus of Nej1 is not only required, but is sufficient, for mediating DNA interactions. Structural characterization revealed that Nej1 exists as a dimer, and that residues 1-244 are sufficient for dimer formation. Examining the ability of this truncated Nej1 (aa 1-244) to perform NHEJ, revealed a defect in end-joining as measured by an in vivo plasmid repair assay. Preliminary functional and structural studies on the Nej1-Lif1 complex suggest that the proteins stably co-purify and the complex binds DNA with a higher affinity than each independent component. The significance of these results is discussed with reference to current literature on Nej1 and other end-joining factors (mammalian and yeast), specifically the recently identified putative mammalian homologue of Nej1, XLF. Collectively, these results demonstrate that although there are several functional similarities, there also appear to be important differences in the structure-function relationships of Nej1 and XLF, and Nej1/XLF and Lif1/Xrcc4.</p> / Thesis / Master of Science (MSc)

De nombreuses études montrent un rôle important de l'architecture du noyau sur la stabilité du génome. Les lamines sont les constituants majeurs de l’enveloppe nucléaire et sont impliquées dans de nombreux processus, notamment, la régulation génique, la réplication et le maintien de la structure du noyau. Il en existe 2 types, les lamines A/C et les lamines B. Certaines mutations des lamines A/C sont à l’origine de syndromes progéroïdes, classés jusqu’à présents en deux catégories : ceux associés à une dérégulation des lamines (laminopathies) et ceux associés à un défaut de réparation de l’ADN, dont l’Ataxie Télangiectasie (A-T). Il est proposé que le vieillissement prématuré observé dans les laminopathies est dû à un défaut de réparation de l’ADN, qui serait alors la voie commune d’induction de sénescence des syndromes progéroïdes. Ceci est appuyé par le fait que de plus en plus de données associent les mutations des lamines A/C à des défauts de réparation de l’ADN. La mise en évidence, par notre laboratoire d’une accumulation de lamine B1 dans A-T et dans deux autres syndromes progéroïdes, pose la question de l’impact de la surexpression de la lamine B1 sur la réparation de l’ADN, d’autant plus que de plus en plus de données associent une augmentation de la lamine B1 à de nombreux cancers, bien que le mécanisme moléculaire ne soit pas connu. Au cours de ma thèse, j’ai donc pu montrer, notamment à l’aide de substrats intra-Chromosomiques, qu’une surexpression de lamine B1 entraînait un défaut de réparation des cassures double-Brin par NHEJ associé à un défaut de recrutement de 53BP1 à la cassure. La mise en évidence d’une interaction entre 53BP1 et la lamine B1, rompue après dommages permet de suggérer un nouveau rôle de la lamine B1 comme réservoir de 53BP1, régulant son recrutement aux cassures. De plus, d’autres résultats suggèrent que la lamine B1 agirait également au niveau de la signalisation du dommage en altérant l’activation d’ATM par un mécanisme qu’il reste à caractériser. L’ensemble de ces résultats montrent un nouveau rôle très important de la lamine B1 dans la signalisation des dommages et la régulation du recrutement des protéines de réparation, ouvrant la voie à une meilleure compréhension de l’implication de la lamine B1 dans la sénescence et le cancer. / Many studies show an important role of nuclear shape on genome stability. Lamins are the major components of the nuclear envelope and are implicated in numerous processes like gene regulation, DNA replication and the maintenance of nuclear structure. There are 2 types of lamins : lamin A/C and lamin B. Some mutations of lamin A/C cause progeroid syndromes, which are classified untill now in two categories : those due to lamins deregulation and those due to DNA repair defects, including Ataxia Telangiectasia (A-T). Accelerated aging observed in laminopathies is proposed to be due to a DNA repair defect, which would be the common pathway leading to senescence in progeroid syndromes. This is supported by many data linking lamin A mutations to DNA repair defects. Our laboratory reported that lamin B1 accumulates in A-T and Fanconi and another study showed also an accumulation in Werner syndrome, which is another progeroïd syndrome. This discovery raises a question about the impact of lamin B1 overexpression on DNA repair, especially as more and more data show an increase of lamin B1 in several cancers, although the molecular mechanism is still unclear. During my thesis, I showed, in particular with intrachromosomal substrates, that lamin B1 overexpression leads to an NHEJ double-Strand break (DSB) repair defect associated with a defect of 53BP1 recruitment to the break. The discovery of an interaction between 53BP1 and lamin B1, which is broken after damage, suggests a new role of lamin B1 as a « reservoir » of 53BP1, regulating its recruitment to the break. In addition, we obtained results suggesting that lamin B1 could also act in the DSB signalisation pathway by affecting ATM activation through a mechanism that we still have to characterize.All together, these datas show a new important role of lamin B1 in DSB signalisation and in the regulation of the recruitment of repair proteins, paving the way to a better understanding of the implication of lamin B1 in senescence and cancer.

The type 1 insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that mediates diverse cellular functions including growth, differentiation, migration and apoptosis protection. IGF-1R signalling has been implicated in tumorigenesis in a variety of cancers, and IGF-1R inhibitory drugs are currently undergoing clinical evaluation. Previous work in our laboratory has shown IGF-1R over-expression in urological cancers at both the mRNA and protein level, thus making it a potential therapeutic target. The first aim of this project was to develop a protocol for IGF-1R immunohistochemistry, investigate the expression and cellular distribution of the IGF-1R receptor in clear cell renal cell carcinomas (ccRCC), and assess correlation with clinical parameters. In tissue microarray analysis, IGF-1R was detected in ~90% of 195 ccRCCs, with signal in the plasma membrane, cytoplasm and also in the nucleus. The presence of nuclear IGF-1R in up to 50% of ccRCCs and its association with adverse prognosis was a novel finding, and suggests that nuclear IGF-1R may influence ccRCC biology. Further investigations will clarify its role in the nucleus and its potential as a prognostic biomarker. The second aim was to investigate effects of IGF-1R inhibition on radiosensitivity and DNA repair, following previous work in our laboratory showing that IGF-1R depletion enhances chemo- and radio-sensitivity, delays double strand break (DSB) resolution, and may play a role in the homologous recombination (HR) pathway of DNA DSB repair. However, the repair defect seen in these early experiments was larger than could be entirely explained by a defect in HR. The current project used a small molecule IGF-1R tyrosine kinase inhibitor AZ12253801 (AstraZeneca), which blocked IGF-1 induced IGF-1R activation and inhibited cell survival. AZ12253801 enhanced the radiosensitivity of prostate cancer cells, which appeared to be independent of effects of IGF-1R inhibition on cell cycle distribution and apoptosis induction. IGF-1R inhibition delayed the resolution of γH2AX foci, supporting a potential role for the IGF-1R in DSB repair. This delay in focus resolution was apparent at early time-points (less than 4 hr), and was epistatic with DNA dependent protein kinase (DNAPK) inhibition in prostate cancer cells and DNAPK deficiency in glioblastoma cells. These results suggest a role for the IGF-1R in the non-homologous end-joining (NHEJ) pathway of DNA DSB repair. A cell-based reporter assay in HEK-293 cells confirmed that IGF-1R inhibition suppressed DSB repair by NHEJ, helping to explain the radiosensitization demonstrated upon IGF-1R inhibition. There was lack of support for a transcriptional effect, with no significant change observed in gene expression on microarray analysis. Although the mechanism of this effect remains unclear, the observed inhibition of NHEJ has implications for the use of IGF-1R inhibitors in combination with DNA damaging agents in cancer treatment.

Abstract The maintenance of integrity of the genome is essential for a cell. DNA repair and faithful DNA replication ensure the stability of the genome. DNA polymerases (pols) are the enzymes that synthesise DNA, a process important both in DNA replication and repair. In DNA replication DNA polymerases duplicate the genome during S phase prior to cell division. Pols α, δ, and ε are implicated in chromosomal DNA replication, but their exact function in replication is not yet completely clear. The mechanisms of different repair pathways and proteins involved are not yet completely characterised either. The deeper understanding of DNA repair and replication mechanisms is crucial for our understanding on the function of the cell. The mechanism of repair of DNA double strand breaks (DSBs) by non-homologous end joining (NHEJ) was studied with an in vitro assay. DNA polymerase activity was found to be involved in NHEJ and important in stabilising DNA ends. Antibodies against pol α, but not pol β or ε, decreased NHEJ significantly, which indicates the involvement of pol α in NHEJ. In addition, the removal of proliferating cell nuclear antigen (PCNA) slightly decreased NHEJ activity. The division of labour between pols α, δ, and ε during DNA replication was studied. Results from UV-crosslinking, chromatin association, replication in isolated nuclei, and immunoelectron microscopy (IEM) studies showed that there are temporal differences between the activities and localisations of the pols during S phase. Pol α was active throughout S phase, pol ε was more active at early S phase, whereas the activity of pol δ increased as S phase advanced. These results suggest that pols δ and ε function independently during DNA replication. Pol ε could be crosslinked to nascent RNA, and this labelling was not linked to DNA replication, but rather to transcription. Immunoprecipitation studies indicated that pol ε, but not pols α and δ, associated with RNA polymerase II (RNA pol II). Only the hyperphosphorylated, transcriptionally active RNA pol II was found to associate with pol ε. A large proportion of pol ε and RNA pol II colocalised in cells as determined with immunoelectron microscopy. The interaction between pol ε and RNA pol II suggests that they are involved in a global regulation of transcription and DNA replication.

Abstract Analysis of the primary structure of DNA polymerase ε B subunit defined similarities to B subunits of eukaryotic DNA polymerases α, δ and ε as well as the small subunits of DNA polymerase DI of Euryarchaeota. Multiple sequence alignment of these proteins revealed the presence of 12 conserved motifs and defined a novel protein superfamily. The members of the B subunit family share a common domain architecture, suggesting a similar fold, and arguing for a conserved function among these proteins. The contribution of human DNA polymerase ε to nuclear DNA replication was studied using the antibody K18 that specifically inhibits the activity of this enzyme in vitro. This antibody significantly inhibited DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei, but did not inhibit SV40 DNA replication in vitro. These results suggest that the human DNA polymerase ε contributes substantially to the replicative synthesis of DNA and emphasises the differences between cellular replication and viral model systems. The human DNA polymerases ε and δ were found capable of gap-filling DNA synthesis during nucleotide excision repair in vitro. Both enzymes required PCNA and the clamp loader RFC, and in addition, polymerase δ required Fen-1 to prevent excessive displacement synthesis. Nucleotide excision repair of a defined DNA lesion was completely reconstituted utilising largely recombinant proteins, only ligase I and DNA polymerases δ and ε provided as highly purified human enzymes. This system was also utilised to study the role of the transcription factor II H during repair. Human non-homologous end joining of model substrates with different DNA end configurations was studied in HeLa cell extracts. This process depended partially on DNA synthesis as an aphidicolin-dependent DNA polymerase was required for the formation of a subset of end joining products. Experiments with neutralising antibodies reveal that DNA polymerase α but not DNA polymerases β or ε, may represent this DNA polymerase activity. Our results indicate that DNA synthesis contributes to the stability of DNA ends, and influences both the efficiency and outcome of the end joining event. Furthermore, our results suggest a minor role of PCNA in non-homologous end joining.

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.