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Anbalagan et. al., PLoS Genet. 2011 March; 7(3): e1002024 Figure 6: RIF1 deletion enhances ssDNA formation at native telomeres of cdc13 cells. Figure 6: (A) Wild type and otherwise isogenic cdc13-1, cdc13-1 rif1Δ and rif1Δ cell cultures exponentially growing at 20°C (lanes 1–4) were incubated at 25°C for 3 hours (lanes 5–8). Wild type and otherwise isogenic cdc13-5 and cdc13-5 rif1Δ cell cultures were grown exponentially at 25°C (lanes 9–11). Genomic DNA was digested with XhoI and single-stranded telomere overhangs were visualized by in-gel hybridization (native gel) using an end-labelled C-rich oligonucleotide. The same DNA samples were separated on a 0.8% agarose gel, denatured and hybridized with the end-labeled C-rich oligonucleotide for loading and telomere length control (denatured gel). (B) Cell cultures were arrested in G2 with nocodazole at 20°C (right) and then transferred at 25°C in the presence of nocodazole for 3 hours (left), followed by analysis of singlestranded telomere overhangs (top) as in panel A, and FACS analysis of DNA content (bottom). 72
Anbalagan et. al., PLoS Genet. 2011 March; 7(3): e1002024 Because the length of single-stranded G overhangs increases during S phase [8], the strong telomeric ssDNA signals observed in cdc13-1 rif1Δ cell cultures at 25°C (Figure 6A) might be due to an enrichment of S/G2 cells. We ruled out this possibility by monitoring the levels of single-stranded TG sequences in cdc13-1 rif1Δ cell cultures that were arrested in G2 with nocodazole at 20°C and then transferred to 25°C in the presence of nocodazole for 3 hours (Figure 6B). Similarly to what we observed in exponentially growing cell cultures, G2arrested cdc13-1 rif1Δ cells at 20°C displayed increased amounts of ssDNA compared to each single mutant under the same conditions, and incubation at 25°C led to further increase of this ssDNA (Figure 6B). Taken together, these findings indicate that the lack of Rif1 causes a severe defect in telomere protection when Cdc13 activity is partially compromised. The lack of Exo1 counteracts DNA damage checkpoint activation and telomeric ssDNA accumulation in cdc13 rif1Δ cells If telomeric ssDNA accumulation contributes to checkpoint activation in cdc13- 1 rif1Δ and cdc13-5 rif1Δ cells, then mutations reducing ssDNA generation should alleviate the arrest and relieve the lethality caused by the lack of Rif1 in cdc13-1 and cdc13-5 background. Because the Exo1 nuclease contributes to generate telomeric ssDNA in cdc13-1 cells [47], we examined the effect of deleting EXO1in cdc13 rif1Δ cells. When G2-arrested cell cultures at 20°C were transferred to 25°C for 3 hours, cdc13-1 rif1Δ exo1Δ triple mutant cells contained significantly lower amounts of telomeric ssDNA than cdc13-1 rif1Δ cells (Figure 6B). A similar behaviour of the triple mutant was detectable even when G2-arrested cultures where kept at 20°C, although the quantity of telomeric ssDNA accumulated by cdc13-1 rif1Δ cells at this temperature was lower than at 25°C (Figure 6B). Furthermore, EXO1 deletion partially suppressed both the temperature-sensitivity of cdc13-1 rif1Δ cells (Figure 7A) and the loss of viability ofcdc13-5 rif1Δ cells (Figure 7B), further supporting the hypothesis that reduced viability in these strains was due to defects in telomere protection. 73
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UNIVERSITÀ DEGLI STUDI DI MILANO-B
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Contents
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Abstract
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Riassunto
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Introduction
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Introduction In Saccharomyces cerev
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Introduction Figure 3: A: Diagram s
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Introduction The Replication Protei
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Introduction Rap1 C terminal is ess
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Introduction and highest at G1 phas
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Introduction cleaves the RNA strand
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Introduction In the G1 cell cycle p
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Introduction impaired, and nuclease
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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
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
Page 79: Anbalagan et. al., PLoS Genet. 2011
Page 95 and 96: References Anbalagan et. al., PLoS
Page 99: Anbalagan et. al., PLoS Genet. 2011
Page 102 and 103: Discussion The DNA damage proteins
Page 104 and 105: Discussion An hypothesis for the sp
Page 106 and 107: Discussion Figure 14: Model, part 2
Page 108 and 109: Telomeric G4 Discussion Apart from
Page 110 and 111: References 1. Hayflick L (1965) The
Page 112 and 113: References that is required for tel
Page 114 and 115: References 51. Surovtseva YV, Churi
Page 116 and 117: References telomere elongation in S
Page 118 and 119: References 100. Walker JR, Corpina
Page 120 and 121: References 124. Gravel S, Larrivée
Page 122 and 123: References 147. Lamarche BJ, Orazio
Page 124 and 125: References 170. Boltz KA, Leehy K,
Page 126 and 127: References 194. Nimonkar AV, Ozsoy
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