Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the �end replication problem�. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the �Telomere Hypothesis of Senescence�. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.

Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the �end replication problem�. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the �Telomere Hypothesis of Senescence�. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.

In normal human somatic cells, the length of telomeres (chromosomal end structures) decreases with each cell division until reaching a critically short length, which halts cell proliferation and induces senescence. The enzyme telomerase, which functions to maintain telomeres at a length that is permissive for cell division, is expressed in approximately 85% of cancer cells and some stem and progenitor cells, including haematopoietic progenitor cells (HPCs), but not most other normal somatic cells. Previous investigations have demonstrated that ectopic expression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity, resulting in telomere elongation in some normal human cell types. However, similar experiments performed in HPCs and endothelial cells have demonstrated a dissociation between the expression of telomerase activity and telomere lengthening. This thesis is focussed on further investigating telomerase-mediated telomere length regulation in HPCs and endothelial cells. Short telomeres in bone marrow and blood leukocytes are associated with the development of disorders linked to bone marrow failure. However, to date a relationship between telomere length and myeloid cell proliferative potential has not been demonstrated. In the current investigations, the telomere length and proliferative potential of 31 cord blood-derived HPCs was determined. Regression analysis revealed a significant correlation between mean telomere length and erythroid cell expansion, but not expansion of other myeloid lineage cells. Another novel finding was that telomerase activity was upregulated in lineage-committed CD34- erythroid cells that were positive for the erythroid-specific lineage marker glycophorin A. It was also functionally demonstrated that telomerase activity facilitates the maximum expansion of erythroid cells. To address the dissociation between telomerase activity and telomere maintenance in BMECs, a dominant negative mutant of the telomere binding protein TRF1, which functions to regulate telomere accessibility, was over-expressed in hTERT-transduced BMECs. These studies showed that telomere access, as well as oncogene expression and exposure to oxidative stress, contribute to telomere length regulation in BMECs. Overall, the results from these investigations demonstrate for the first time the functional significance of telomere length and telomerase activity in ex vivo expansion of erythroid cells, and provide novel insight to the molecular complexity of telomere length maintenance in endothelial cells.

Telomeres are important protein-DNA structures at the ends of linear eukaryotic chromosomes that are necessary to prevent chromosome fusions and exonuclease attack. We found that telomere tracts in Arabidopsis are fairly uniformly distributed throughout a size range of 2-9kb. Unexpectedly, telomeres in WS plants displayed a bimodal size distribution with some individuals exhibiting 4-8 kb telomeres and others 2-5 kb telomeres. We also examined the dynamics of telomere tracts on individual chromosome ends. Following the fate of telomeres in plants through successive generations, we found that the shortest telomeres were typically elongated in the subsequent generation, while the longest telomeres were usually shortened. Thus, telomere length homoeostasis is achieved through intermittent telomerase action on shorter telomeres to attain an optimal size.Single-strand telomere binding proteins were also analyzed. Four major telomere binding protein complexes from cauliflower were identified and their DNA-binding properties characterized. The DNA-binding component of one of the complexes was purified and analyzed by mass-spectrometry. Peptide mass data was used to search for putative protein candidates from the Arabidopsis thaliana database. Additionally, two Arabidopsis genes, AtPot1 and AtPot2, were identified and characterized. The genes encode two single-strand telomeric DNA binding proteins. AtPot1 and AtPot2 proteins can homo- and heterodimerize in vitro. Pot1 protein predominantly localizes to the nucleolus, whereas Pot2 is exclusively nuclear. Plants over-expressing full-length Pot1 and Pot2 proteins had no obvious phenotype, while over-expression of P2DBD and P1∆DBD caused moderate telomere shortening. Plants over-expressing P2DBD had severe morphological and reproductive defects, multiple chromosome abnormalities and aneuploidy. Over-expression of a chimeric protein DBD-P1∆DBD led to rapid telomere shortening, confirming the involvement of Arabidopsis Pot proteins in telomere length maintenance. Intriguingly, telomerase in DBD-P1∆DBD-EYFP plants is inactivated, suggesting that Pot proteins are also involved in regulation of telomerase activity. The analysis of Arabidopsis telomeres and telomere binding proteins will provide additional information towards understanding the role of the telomeric nucleoprotein complex in eukaryotic chromosome biology.

Telomeres comprise the physical ends of chromosomes. In the absence of telomerase, the enzyme responsible for replenishing telomeric DNA, telomeres progressively shorten due to the end replication problem. Eventually telomeres reach a length where they are recruited into end-to-end chromosome fusions. Through the use of novel PCR strategies, I followed the fate of telomeres in plants lacking telomerase as they progressed into dysfunction. I uncovered two distinct structural/functional length transitions. The first transition (~1 kb) marks the onset of telomere dysfunction, where telomeres are transiently uncapped and a subset of them engage in end-to-end fusions. The second transition (~300 bp) defines complete telomere dysfunction as telomeres below this length lack G-overhangs and the vast majority of the chromosome ends fuse. Thus, these two telomere lengths define architectural transitions that link structure and function. In addition, I uncovered a hierarchy of end-joining pathways that join dysfunctional telomeres in which the non-homologous end-joining (NHEJ) protein, KU predominates. In the absence of KU, telomeres are joined by a microhomologymediated end-joining pathway (MMEJ) that is dependent on Mre11. I also show that DNA ligase IV (LIG4) is the predominant enzyme that ligates dysfunctional telomeres as fusions are reduced in its absence. These studies highlight the importance of repairing DSBs and demonstrate that Arabidopsis possesses highly redundant means for processing dysfunctional telomeres. The G-overhang is an essential feature of the telomere that is required for proper telomere function. I employed methods to examine G-overhang status in various mutants known to contribute to telomere maintenance in Arabidopsis. My analysis revealed that the putative G-overhang binding proteins POT1a, POT1b and POT1c, make modest, but distinct contributions to the G-overhangs. Additionally, I uncovered a major role for the putative telomere capping protein, CIT1 in maintenance of the Goverhang. G-overhang signals obtained from cit1 mutants were grossly increased indicating that CIT1 is involved in either protecting the C-rich strand of the telomere from nuclease attack, or in controlling telomerase extension of the G-strand. Together, these data have provided new insight into factors that contribute to telomere integrity and have further developed Arabidopsis as a model for telomere biology.

Telomeres comprise the physical ends of chromosomes. Essential functions of telomeres include protecting the terminus from being recognized as a DNA doublestrand break and facilitating the complete replication of the physical end of the DNA. Telomere functions are mediated by a large array of telomere-associated proteins. Mutations in telomere-related genes cause immediate telomere dysfunction, activation of DNA damage response, and accumulation of end-to-end chromosome fusions. In addition, changes in telomere complex composition may affect the ability of the telomerase enzyme to maintain telomeres in vivo. Here, we describe the characterization of telomere-associated proteins in the flowering plant, Arabidopsis thaliana. Using a bioinformatics approach, we identified twelve proteins with sequence similarity to vertebrate duplex telomere DNA binding proteins TRF1 and TRF2. We showed that, like their vertebrate counterparts, some of the Arabidopsis TRFL (TRF-LIKE) proteins can homodimerize and bind telomeric DNA in vitro, indicating that Arabidopsis encodes a large family of double-strand telomeric DNA binding proteins. We have also characterized three Arabidopsis POT1 proteins whose homologs in yeast and vertebrates associate with the single-stranded portion of telomeric DNA. Unexpectedly, we found that unlike POT1 protein in other organisms, Arabidopsis AtPOT1a protein associates with telomeres only in the S phase of the cell cycle and is a physical component of the active telomerase RNP complex, providing positive telomere length regulation. Our data implicated AtPOT1b, another Arabidopsis POT1 protein, in chromosome end protection. Finally, we showed that Arabidopsis thaliana has evolved a third POT1 protein, AtPOT1c, which contributes to both telomere length regulation and telomerase activity, and maintenance of the structure of the chromosome terminus. Thus, Arabidopsis has evolved a set of POT1 proteins that make distinct and novel contributions to telomere biology. Finally, we describe the identification and characterization of a novel Arabidopsis protein CIT1 (Critical for Integrity of Telomeres 1), and show that CIT1 deficiency leads to an immediate and profound telomere dysfunction and chromosome end deprotection. Altogether, these data provide new insight into plant telomereassociated factors and significantly improve our understanding of the overall architecture and evolution of telomeric complex in Arabidopsis.

Telomeres are important protein-DNA structures at the ends of linear eukaryotic chromosomes that are necessary to prevent chromosome fusions and exonuclease attack. We found that telomere tracts in Arabidopsis are fairly uniformly distributed throughout a size range of 2-9kb. Unexpectedly, telomeres in WS plants displayed a bimodal size distribution with some individuals exhibiting 4-8 kb telomeres and others 2-5 kb telomeres. We also examined the dynamics of telomere tracts on individual chromosome ends. Following the fate of telomeres in plants through successive generations, we found that the shortest telomeres were typically elongated in the subsequent generation, while the longest telomeres were usually shortened. Thus, telomere length homoeostasis is achieved through intermittent telomerase action on shorter telomeres to attain an optimal size.Single-strand telomere binding proteins were also analyzed. Four major telomere binding protein complexes from cauliflower were identified and their DNA-binding properties characterized. The DNA-binding component of one of the complexes was purified and analyzed by mass-spectrometry. Peptide mass data was used to search for putative protein candidates from the Arabidopsis thaliana database. Additionally, two Arabidopsis genes, AtPot1 and AtPot2, were identified and characterized. The genes encode two single-strand telomeric DNA binding proteins. AtPot1 and AtPot2 proteins can homo- and heterodimerize in vitro. Pot1 protein predominantly localizes to the nucleolus, whereas Pot2 is exclusively nuclear. Plants over-expressing full-length Pot1 and Pot2 proteins had no obvious phenotype, while over-expression of P2DBD and P1∆DBD caused moderate telomere shortening. Plants over-expressing P2DBD had severe morphological and reproductive defects, multiple chromosome abnormalities and aneuploidy. Over-expression of a chimeric protein DBD-P1∆DBD led to rapid telomere shortening, confirming the involvement of Arabidopsis Pot proteins in telomere length maintenance. Intriguingly, telomerase in DBD-P1∆DBD-EYFP plants is inactivated, suggesting that Pot proteins are also involved in regulation of telomerase activity. The analysis of Arabidopsis telomeres and telomere binding proteins will provide additional information towards understanding the role of the telomeric nucleoprotein complex in eukaryotic chromosome biology.

Gene silencing in Saccharomyces cerevisiae has been conclusively linked to histone deacetylation and the subsequent formation of heterochromatin. The participating histone deacetylases have also been well characterized. In contrast, the opposing histone acetyltransferases (HAT) and their mechanism of action remain elusive. In particular, very little is known about the effects of two of the nine HATs in S. cerevisiae, ESA1 and RTT109. The focus of my research was to test if these HATs influence the silencing of genes at the subtelomeric regions of S.cerevisiae and to assess their mechanism of action. To address these issues I used a panel of recombinant telomeric constructs that harbor the URA3 reporter gene. These constructs were inserted at the VIIL telomere of the mutant strains esa1-414 and ∆rtt109. The level of gene repression of the URA3 reporter in each construct in both of these strains has been assessed by a routine assay measuring the sensitivity of the strain to the drug 5-fluoro-orotic acid. My results indicate than none of these HATs plays a specific major role in gene repression at telomeres. Instead, they show general anti-silencing activity that cannot be linked to any specific sub-telomeric elements. Like many other HATs, Esa1 and Rtt109 seem to operate through global acetylation of histones rather than through specific recruitment. These results provide additional insight in the function of the HATs in gene silencing and suggest that there are multiple mechanisms that we do not yet understand. The information provided here would help in future studies to determine the mode of action of anti-silencers and subtelomeric elements involved in telomeric silencing. These issues are of fundamental significance and will contribute to the dynamic and expanding field of epigenetics. / NSERC 400478

In normal human somatic cells, the length of telomeres (chromosomal end structures) decreases with each cell division until reaching a critically short length, which halts cell proliferation and induces senescence. The enzyme telomerase, which functions to maintain telomeres at a length that is permissive for cell division, is expressed in approximately 85% of cancer cells and some stem and progenitor cells, including haematopoietic progenitor cells (HPCs), but not most other normal somatic cells. Previous investigations have demonstrated that ectopic expression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity, resulting in telomere elongation in some normal human cell types. However, similar experiments performed in HPCs and endothelial cells have demonstrated a dissociation between the expression of telomerase activity and telomere lengthening. This thesis is focussed on further investigating telomerase-mediated telomere length regulation in HPCs and endothelial cells. Short telomeres in bone marrow and blood leukocytes are associated with the development of disorders linked to bone marrow failure. However, to date a relationship between telomere length and myeloid cell proliferative potential has not been demonstrated. In the current investigations, the telomere length and proliferative potential of 31 cord blood-derived HPCs was determined. Regression analysis revealed a significant correlation between mean telomere length and erythroid cell expansion, but not expansion of other myeloid lineage cells. Another novel finding was that telomerase activity was upregulated in lineage-committed CD34- erythroid cells that were positive for the erythroid-specific lineage marker glycophorin A. It was also functionally demonstrated that telomerase activity facilitates the maximum expansion of erythroid cells. To address the dissociation between telomerase activity and telomere maintenance in BMECs, a dominant negative mutant of the telomere binding protein TRF1, which functions to regulate telomere accessibility, was over-expressed in hTERT-transduced BMECs. These studies showed that telomere access, as well as oncogene expression and exposure to oxidative stress, contribute to telomere length regulation in BMECs. Overall, the results from these investigations demonstrate for the first time the functional significance of telomere length and telomerase activity in ex vivo expansion of erythroid cells, and provide novel insight to the molecular complexity of telomere length maintenance in endothelial cells.

TRF2 is a multifunctional protein implicated in telomere length maintenance, DNA double strand break repair and telomere protection. TRF2 undergoes extensive post-translational modification including phosphorylation. Mass spectrometry analysis has identified two candidate TRF2 phosphorylation sites: T317 and S323. In this study, the roles of these two potential phosphorylation sites were examined for their role in cell growth, telomere length maintenance and DNA damage response. Through retroviral infection, HT1080, HeLaII and GM847 cell lines stably expressing the vector alone, Myc-tagged wild type TRF2, Myc-tagged TRF2 carrying a nonphosphorylatable mutation of either T317A or S323A and Myc-tagged TRF2 carrying a phosphomimic mutation of either T317D or S323D were generated. Overexpression of TRF2 mutant alleles has no effect on cell growth and proliferation as well as TRF2 association with ALT-associated PML bodies. On the other hand, the effect of TRF2 mutant alleles on DNA damage response and telomere length maintenance is inconclusive and requires further investigation. / Thesis / Master of Science (MSc) / TRF2 is a multifunctional protein implicated in telomere length maintenance, DNA damage repair and telomere protection. TRF2 undergoes extensive post-translational modification, which in turn regulates its DNA binding activity, protein stability and cellular localization. TRF2 is found to be phosphorylated at a number of serine/threonine sites, including T317 and S323. In this study, the candidate phosphorylation sites T317 and S323 of TRF2 were analyzed for their potential roles in cell growth, telomere length maintenance and DNA damage response.