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Introduction Marginotomy – End replication problem In 1960’s Leonard Hayflick observed when confluent cells of different origins were continuously subcultured, the cells age and stops dividing after a certain number of cellular divisions. Based on this observation, he hypothesized that aging or senescence might occur at cellular level [1]. Meanwhile, Alexey Matveyevich Olovnikov refined the Watson and Crick’s classical model of DNA replication. Based on Watson and Crick’s model, he expected that with every round of replication the resulting “replica” or daughter strand will be shortened and he called this problem “Marginotomy” (or “End replication problem”) [2]. He proposed two hypotheses: the first one was based on the structural constrain of the DNA polymerase catalytic domain and the second was based on the removal of the RNA primer. Olovnikov very well realized that Marginotomy might be the reason for Hayflick’s observation of limited cellular doubling’s. He hypothesized that the chromosome ends should have some kind of “telo-genes” or “buffer genes”, which could be sacrificed during every successive replication. After the exhaustion of these “telo-genes”, the cell might age or die because it loses essential genes near “telo-genes”. He also hypothesized that cell survival during evolution requires “Anti-Marginotomy”, which can occur when factors regulating Marginotomy are reintroduced into cells and can delay ageing by lengthening “telo-genes”. Overall according to Olovnikov, the terminus of a chromosome was the Achilles Heel and it could be protected by Anti-Marginotomy [2]. Telomeres and telomerase Unaware of Olovnikov’s hypothesis, Elizabeth Blackburn observed that the ends of ciliate Tetrahymena chromosomes are made up of repetitive DNA sequences (3’ strand with T2G4) [3] and few years later the telomere terminal transferase (telomerase enzyme) which can add repetitive DNA was identified. The repetitive nature of the telomeric DNA was indeed the “buffer gene” and telomerase can be considered the Anti-Marginotomy factor hypothesized by Olovnikov [2]. From ciliates to humans, telomeres have conserved features and telomeric sequence are made up of short tandem repeats. Telomeres in different organisms vary by repeat consensus, length, structure and diversity of bound proteins. 3
Introduction In Saccharomyces cerevisiae, telomeres are 300 ± 75 bp of simple repeats (TG1- 3). In the telomeric DNA, the 3’ strand is G-rich and so referred as G-strand, whereas the 5’ C-rich complementary strand is called C-strand. The G-strand extends beyond its complementary C-rich strand to form a single-stranded overhang, referred to as the G-tail [4] (for a detailed review on budding yeast telomeres, please see [5]). Figure 1: Diagram showing the repetitive sequence of telomeric DNA and telomerase reverse transcriptase using RNA template to elongate G-rich telomeric DNA (Source: NobelPrize.org) In budding yeast, EST2 encodes the reverse transcriptase subunit (telomerase). Est2 uses the RNA encoded by the TLC1 gene as a template for telomere elongation [6]. The stem loop of TLC1 RNA serves as scaffold for the binding of Est2, Est1 (telomerase regulatory subunit), Ku heterodimer complex (See DNA damage proteins section for more details) [7–9]. Est1 can bind to telomeric ssDNA independently of its interaction with TLC1 RNA [10]. Cells lacking EST2, TLC1 or EST1 undergo progressive loss of telomeres and senescence [11,12]. Cdc13 binds to telomeric TG1–3 tails using its OB (oligonucleotide/ oligosaccharide binding) domain [13] and interacts with Est1 to promote recruitment and activation of Est2. The strong association of Est2 to telomeres 4
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Page 5 and 6: Abstract
Page 7 and 8: Riassunto
Page 9: Introduction
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 and 32: Introduction Similarly in fission y
Page 33 and 34: Introduction constitutive phosphory
Page 35 and 36: Shelterin-Like Proteins and Yku Inh
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Bonetti et. al., PLoS Genet. 2010 M
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Bonetti et. al., PLoS Genet. 2010 M
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Rif1 Supports the Function of the C
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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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