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Introduction overhangs serve as substrate to telomerase, they are important to maintain telomere length homeostasis. Yeast cells acquire telomeric TG1–3 ss overhang in cell cycle regulated manner [4,199]. The formation of the telomeric G tail and elongation requires the passage of the replication fork at telomeres [14,200]. Cdk1 activity is required for the generation of the long telomeric 3′ overhang in late S phase [70]. Passage of replication fork at telomeres, might require the release of telomere bound proteins and lead to a transient state of telomeric deprotection. At this stage, CDK1-dependent 5′ resection might take place to generate telomeric overhangs. Telomeres must be protected from uncontrolled nucleolytic activities. Telomere shortening caused by telomerase deletion increases the amount of telomeric ssDNA in predominantly Exo1 nuclease dependent manner [201] and triggers a DNA damage and other stress related responses [202]. In yku null cells, telomeres are shorter with accumulation of telomeric ssDNA and checkpoint-mediated cell cycle arrest at elevated temperatures. The generation of telomeric ssDNA in yku70Δ occurs in cell cycle independent manner and due to action of Exo1 nuclease at telomeres [124,131,203]. In cdc13-1 mutant, telomeres undergo Exo1 dependent 5’ C strand degradation and cell cycle arrest at restrictive temperature [31,204]. In contrast to yku lacking cells, cdc13-1 mutant or cells lacking CDC13 or STN1, the overhang generation is cell cycle dependent occurring only in G2/M, but not in G1 of the cell cycle and requires the completion of S phase and Cdk1 kinase activity [205]. This observation supports the hypothesis that requirement of replication fork passage might lead to transient unprotected state at telomeres. Also, normal human telomeres are recognized as DNA damage in G2 phase of cell cycle [206]. At de novo telomeres, MRX complex is involved in telomeric ss Gtail generation at telomeres [138]. MRX is required for generation of proper constitutive telomeric G-tails in linear plasmids [135]. Previous work from our lab discovered that similar to DSB processing, Sae2, Exo1, Sgs1, Dna2 are all involved in generation of telomeric overhangs (See figure 12) [207]. Again, extensive resection of DSB requiring Cdk1 phosphorylation of Sae2, it was also important for telomere overhang formation and telomere elongation. A very interesting finding from this work came from Sae2-S267D variant mimicking 25
Introduction constitutive phosphorylation which did not totally bypass the need of Cdk1 for single-stranded telomeric DNA generation. This hinted that additional Cdk1 targets might be involved in positive or negative regulators of telomere degradation [207]. Figure 12: Similar mechanisms are involved in DNA End Processing at DSBs and Telomeres. Upon phosphorylation of Sae2 by Cdk1, MRX and Sae2 trigger initial resection. Extended overhangs are generated by Sgs1-Dna2. Exo1 can also contribute to overhang formation. Telomere shortening occurs at the leading strand telomere due to end processing. (Source: [208]) As mentioned before, Ku and Cdc13 are important for telomere protection from degradation and lack of Rap1 or Rif2 lead to telomere-telomere fusion due to NHEJ. However, the precise roles of telomere binding proteins in telomere protection at molecular level were still unknown. This Ph.D thesis was aimed at studying how yeast telomeric proteins might protect telomeres from degradation using de novo telomere assay and native in gel hybridization techniques in different cell cycle phases. 26
Page 1 and 2: UNIVERSITÀ DEGLI STUDI DI MILANO-B
Page 3 and 4: Contents
Page 5 and 6: Abstract
Page 7 and 8: Riassunto
Page 9 and 10: Introduction
Page 11 and 12: Introduction In Saccharomyces cerev
Page 13 and 14: Introduction Figure 3: A: Diagram s
Page 15 and 16: Introduction The Replication Protei
Page 17 and 18: Introduction Rap1 C terminal is ess
Page 19 and 20: Introduction and highest at G1 phas
Page 21 and 22: Introduction cleaves the RNA strand
Page 23 and 24: Introduction In the G1 cell cycle p
Page 25 and 26: Introduction impaired, and nuclease
Page 27 and 28: Introduction functionally compromis
Page 29 and 30: Introduction increasing the interac
Page 31: Introduction Similarly in fission y
Page 35 and 36: Shelterin-Like Proteins and Yku Inh
Page 37 and 38: Bonetti et. al., PLoS Genet. 2010 M
Page 65 and 66: Rif1 Supports the Function of the C
Page 67 and 68: Anbalagan et. al., PLoS Genet. 2011
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Anbalagan et. al., PLoS Genet. 2011
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References Anbalagan et. al., PLoS
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Discussion The DNA damage proteins
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Discussion An hypothesis for the sp
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Discussion Figure 14: Model, part 2
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Telomeric G4 Discussion Apart from
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References 1. Hayflick L (1965) The
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References that is required for tel
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References 51. Surovtseva YV, Churi
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References telomere elongation in S
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References 100. Walker JR, Corpina
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References 124. Gravel S, Larrivée
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References 147. Lamarche BJ, Orazio
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References 170. Boltz KA, Leehy K,
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References 194. Nimonkar AV, Ozsoy
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