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Anbalagan et. al., PLoS Genet. 2011 March; 7(3): e1002024 Furthermore, similar amounts of phosphorylated Rad53 were detected in both yku70Δ and yku70Δ rif1Δ cell cultures that were kept at 37°C for 4 hours (Figure 8B), indicating that loss of Rif1 does not enhance the telomere protection defects already present in yku70Δ cells. Consistent with a previous observation [54], RIF1 deletion partially suppressed the temperature sensitivity (Figure 8A) and the telomere length defect (data not shown) caused by the lack of Yku70, suggesting that the elongated state of the telomeres could be the reason why yku70Δ rif1Δ cells can proliferate at 37°C. Checkpoint activation can also be induced during telomere erosion caused by insufficient telomerase activity [55,56]. Thus, we asked whether RIF1 deletion accelerated senescence progression and/or upregulated checkpoint activation in cells lacking the telomerase catalytic subunit Est2. Meiotic tetrads were dissected from a diploid strain heterozygous for the est2Δ and rif1Δ alleles, which are recessive and therefore do not affect telomere length in the diploid. After 2 days of incubation at 25°C (approximately 25 generations), spore clones from the dissection plate were both streaked for 4 successive times (Figure 8C) and propagated in YEPD liquid medium to prepare protein extracts for Rad53 phosphorylation analysis at different time points (Figure 8D). Similar to what was previously observed[44], RIF1 deletion did not accelerate senescence progression in est2Δ cells, as est2Δ rif1Δ clones showed a decline in growth similar to that of est2Δ clones (Figure 8C). Furthermore, est2Δ and est2Δ rif1Δ cell cultures showed similar patterns of Rad53 phosphorylation with increasing number of generations (Figure 8D). Thus, the lack of Rif1 does not enhance either DNA damage checkpoint activation or senescence progression during telomere erosion caused by the lack of telomerase. Finally, because the telomerase machinery is known to be recruited to an unrepaired DSB [57], we ruled out the possibility of a general role for Rif1 in inhibiting checkpoint activation by examining activation/deactivation of the checkpoint induced by an unrepaired DSB. To this end, we used JKM139 derivative strains, where a single DSB can be generated at the MAT locus by expressing the site-specific HO endonuclease gene from a galactose-inducible promoter [58]. This DSB cannot be repaired by homologous recombination, because the homologous donor sequences HML or HMR are deleted. 76
Anbalagan et. al., PLoS Genet. 2011 March; 7(3): e1002024 Figure 8: RIF1 deletion does not influence the checkpoint response to CSTindependent telomere capping deficiencies or to an irreparable DSB. Figure 8: (A) Cell cultures with the indicated genotypes were grown overnight in YEPD at 25°C. Serial 10-fold dilutions were spotted onto YEPD plates and incubated at the indicated temperatures for 2–3 days. (B) Cell cultures exponentially growing in YEPD at 25°C were shifted to 37°C at time 0. Rad53 was visualized at the indicated time points as inFigure 3E. (C) Meiotic tetrads from a EST2/est2ΔRIF1/rif1Δ diploid strain were dissected on YEPD plates. After ~25 generations, spore clones from 20 tetrads were subjected to genotyping and to four successive streak-outs (1X to 4X), corresponding to ~25, ~50, ~75 and ~100 generations of growth, respectively. All tetratype tetrads behaved as the one shown in this panel. (D) After ~25 generations of growth on the dissection plates, est2Δ and est2Δ rif1Δ spore clones were propagated in liquid YEPD medium. At the indicated generations, protein extracts were subjected to western blot analysis with anti-Rad53 antibodies. (E–F) Checkpoint response to an irreparable DSB. (E) YEP+raf G1-arrested cell cultures of wild type JKM139 and its isogenic rif1Δ derivative strain were spotted on galactose-containing plates that were incubated at 28°C (time zero). At the indicated time points, 200 cells for each strain were analyzed to determine the frequency of single cells and of cells forming microcolonies of 2, 4 or more than 4 cells. (F) Galactose was added at time zero to cell cultures of the strains in panel E exponentially growing in YEP+raf. Protein extracts were subjected to western blot analysis with anti-Rad53 antibodies. 77
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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
Page 9 and 10:
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
Page 17 and 18:
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
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Introduction increasing the interac
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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 83: 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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