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The RNA Processing Proteins Xrn1 and Rrp6 Regulate DNA Damage Checkpoint Activation and Telomere Metabolism

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The RNA Processing Proteins Xrn1 and Rrp6 Regulate DNA Damage Checkpoint Activation and Telomere Metabolism SCUOLA DI DOTTORATO UNIVERSITÀ DEGLI STUDI DI MILANO-BICOCCA Dipartimento di Biotecnologie e Bioscienze Dottorato di Ricerca in Scienze della Vita Ciclo XXIX Curriculum in Biotecnologie The RNA processing proteins Xrn1 and Rrp6 regulate DNA damage checkpoint activation and telomere metabolism Cesena Daniele Matricola 718295 Tutore: Prof.ssa Longhese Maria Pia Coordinatore: Prof. Vanoni Marco Ercole ANNO ACCADEMICO 2015/2016 INDEX Index ABSTRACT ................................................................................................... 1 RIASSUNTO ................................................................................................. 6 INTRODUCTION ........................................................................................ 11 Maintaining genome integrity: the DNA Damage Response ............... 13 DNA Double-Strand Breaks (DSBs) ..................................................... 14 DSB repair pathways: Non-Homologous End-Joining (NHEJ) .............. 16 DSB repair pathways: Homologous Recombination (HR) .................... 18 Resection: a crucial step in DSB repair ............................................... 22 Positive regulators of DSB resection .................................................. 23 Negative regulators of DSB resection ................................................. 27 The DNA damage checkpoint ............................................................. 31 Maintaining genome integrity: protection and replication of the natural ends of linear chromosomes.................................................. 37 Capping of Chromosome Ends ........................................................... 40 End processing at telomeres .............................................................. 44 mRNA degradation pathways: deadenylation-dependent decay ........ 50 mRNA degradation pathways: deadenylation-independent and endonucleolytic cleavage-dependent decay ...................................... 54 Maintaining genome integrity: a role for RNA processing .................. 58 RESULTS .................................................................................................... 63 RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. ........................................................ 65 Xrn1, Rrp6 and Trf4 are necessary for Mec1/ATR activation in response to a DSB............................................................................................. 67 Xrn1 promotes resection of DNA ends ............................................... 72 Xrn1 supports MRX function in DSB resection .................................... 73 Rrp6 and Trf4 promote the loading of RPA and Mec1 to the DSB ....... 78 Rrp6 and Trf4 are not required for HR repair of a DSB ....................... 82 The lack of Xrn1, Rrp6 or Trf4 does not affect expression of most DDR genes ................................................................................................ 85 Index Regulation of telomere metabolism by the RNA processing protein Xrn1 ............................................................................................................. 94 The lack of Xrn1 or Rrp6 partially suppresses the temperature sensitivity of cdc13-1 cells ................................................................. 99 Xrn1 and Rrp6 are required to fully activate the checkpoint at uncapped telomeres........................................................................ 102 Xrn1 and Rrp6 regulate telomere capping through a mechanism that is distinct from that involving the NMD pathway ................................ 103 Xrn1 is required to generate ssDNA at uncapped telomeres ............ 108 Xrn1 maintains telomere length by acting as a cytoplasmic nuclease ........................................................................................................ 111 Xrn1 promotes Cdc13 association to telomeres independently of ssDNA generation ............................................................................ 116 Xrn1 promotes Cdc13 association at telomeres by downregulating Rif1 level ................................................................................................ 122 DISCUSSION ............................................................................................ 127 MATERIALS AND METHODS .................................................................... 137 Yeast and bacterial strains .................................................................. 139 Yeast strains and plasmids ............................................................... 139 E. coli strain ..................................................................................... 140 Growth media..................................................................................... 145 S. cerevisiae media .......................................................................... 145 E. coli media .................................................................................... 146 Conservation and storage of S. cerevisiae and E. coli strains ............ 146 Molecular biology techniques ............................................................ 147 Agarose gel electrophoresis ............................................................. 147 DNA extraction from agarose gels (paper strip method) .................. 148 Restriction endonucleases ............................................................... 148 Ligation ........................................................................................... 149 Polymerase Chain Reaction (PCR) .................................................... 149 Preparation of yeast genomic DNA for PCR ...................................... 151 Index Plasmid DNA extraction from E. coli (I): minipreps boiling ................ 151 Plasmid DNA extraction from E. coli (II): minipreps with QIAGEN columns .......................................................................................... 151 Transformation of E. coli DH5α ........................................................ 152 Transformation of S. cerevisiae ........................................................ 152 Extraction of yeast genomic DNA (Teeny yeast DNA preps) ............. 153 Southern blot analysis ..................................................................... 153 Southern blot analysis of telomere length ....................................... 154 In-Gel Hybridization ......................................................................... 154 DSB end resection ........................................................................... 155 DSB repair ....................................................................................... 155 qRT-PCR .......................................................................................... 155 Total RNA-Seq analysis .................................................................... 156 Sub-cellular fractionation ................................................................ 157 Chromatin ImmunoPrecipitation (ChIP) analysis .............................. 158 Coimmunoprecipitations ................................................................. 160 Synchronization of yeast cells ............................................................. 161 Synchronization of yeast cells with α-factor ..................................... 161 Synchronization of yeast cells with nocodazole................................ 161 Other techniques ................................................................................ 162 FACS analysis of DNA contents ........................................................ 162 Total protein extracts ...................................................................... 162 SDS-PAGE and western blot analysis ................................................ 163 Drop test ......................................................................................... 164 Fluorescence microscopy................................................................. 164 REFERENCES ........................................................................................... 167 ABSTRACT Abstract Genome instability is one of the most pervasive characteristics of cancer cells. It can be due to DNA repair defects, failure to arrest the cell cycle and loss of telomere-end protection that lead to end-to-end fusion and degradation. Among the many types of DNA damage, the DNA Double Strand Break (DSB) is one of the most severe, because it can cause mutations and chromosomal rearrangements. Eukaryotic cells respond to DSBs by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR, in order to arrest the cell cycle until DSBs are repaired. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA) that arises upon nucleolytic degradation (resection) of the DSB. A similar checkpoint response is triggered when the natural ends of eukaryotic chromosomes lose their protection, resembling and being recognized as DSBs. This protection is provided by specialized nucleoprotein complexes called telomeres. Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3’-ended single- stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidences indicate that RNA processing proteins play critical, yet poorly understood, roles in genomic stability and telomere metabolism. We
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