ausblenden:
Sprache(n):
eng - English
Datum:
2004-07-13
Publikationsstatus:
Angenommen
Seiten:
96 pp
Ort, Verlag, Ausgabe:
Berlin : Humboldt-Universität
Inhaltsverzeichnis:
1 Introduction 1
1.1 N-terminal acetylation of proteins 1
1.2 Nα-acetyltransferases in S. cerevisiae 3
1.3 NatA – the major Nα-acetyltransferase complex
of S. cerevisiae 5
1.4 Chromatin and gene 8
1.5 Chromatin modifying processes 11
1.6 Silencing in S. cerevisiae 14
1.7 Silencing proteins investigated in this thesis 22
1.8 Outline of this thesis 25
2 Materials and methods 28
2.1 Materials 28
2.1.1 E. coli strains 28
2.1.2 Yeast strains 28
2.1.3 Growth conditions and media 30
2.1.4 Plasmid constructions 31
2.1.5 Oligonucleotides 32
2.1.6 Buffers 32
2.2 Methods 32
2.2.1 Yeast strain construction 32
2.2.2 Molecular cloning techniques 34
2.2.3 Silencing assays 35
2.2.4 Two-hybrid assay 36
2.2.5 Immunofluorescence on yeast cells 36
2.2.6 Biochemical techniques 36
3 Results 39
3.1 Nat1 was required for repression of the HM loci, telomeres and the rDNA locus 39
3.2 Orc1 required Nα-acetylation by NatA for its function in telomeric silencing 40
3.2.1 Tethering of Orc1 or Sir1 to the silencer bypassed the requirement for NatA in silencing 40
3.2.2 Orc1 was N-terminally acetylated by NatA 42
3.2.3 Unacetylated orc1 mutants displayed telomeric derepression 43
3.2.4 HM silencing was not affected by the lack of N-terminal acetylation of Orc1 47
3.2.5 Nα-acetylation was not required for the protein stability of Orc1 47
3.2.6 NatA activity, but not Nα-acetylation of Orc1, was required for replication 48
3.2.7 Synthetic lethality between nat1∆ and SUM1-1 was suppressed by orc1∆1-235 49
3.3 N-terminal deletions of Orc1 caused silencing defects distinct from those of nat1∆ 50
3.3.1 HMR silencing was disrupted in N-terminally truncated orc1 mutants 50
3.3.2 Alpha-factor sensitivity was reduced in N-terminally truncated orc1 mutants 52
3.3.3 N-terminal truncations of Orc1 enhanced the α-factor resistance of sir1∆ 53
3.3.4 Telomeric silencing was affected by N-terminal truncations of Orc1 54
3.3.5 Replication was not disturbed by N-terminal truncations of Orc1 55
3.3.6 The N-terminal 51 amino acids of Orc1 were required for its two-hybrid interaction with Sir1 55
3.4 Sir3 was a substrate of NatA 56
3.4.1 Sir3 was Nα-acetylated by NatA 56
3.4.2 NatA activity was required to localize Sir3 to perinuclear foci 57
3.5 A genetic screen for multicopy suppressors of the nat1∆ silencing defect 58
3.5.1 Screening for restored silencing of HMR SS ∆I in a nat1∆ strain 58
3.5.2 Overexpression of SSF2 suppressed the nat1∆ mating defect 60
3.5.3 Overexpression of ORC1 did not suppress the mating defect caused by nat1∆ 61
4 Discussion 63
4.1 Relevance of Nα -acetylation for Orc1 63
4.2 Function of the N-terminal 100 amino acid domain of
Orc1 66
4.3 A model of the role of NatA in silencing 69
4.4 Nα-acetylation as a conserved eukaryotic protein modification 71
References 73
Abbreviations 92
Curriculum vitae 93
Publications 94
Acknowledgements 95
Art der Begutachtung:
-
Identifikatoren:
eDoc: 226112
Art des Abschluß:
Doktorarbeit
