Understanding the function of the Rtt101 E3 ubiquitin ligase in response to replication stress Dissertation zur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie der Johannes Gutenberg-Universität Mainz vorgelegt von Vanessa Kellner geboren am 23.09.1988 in Mannheim, Germany Mainz, 2018 Tag der mündlichen Prüfung: 18. Mai 2018 List of Publications Table of Contents List of Publications .................................................................................................. V Summary ................................................................................................................. VII Zusammenfassung .................................................................................................. IX 1. Introduction ........................................................................................................... 1 1.1 Ubiquitylation – mechanism and functions ........................................................ 1 1.2 Ubiquitylation in DNA replication and repair ...................................................... 3 1.3 The Cullin E3 ubiquitin ligase Rtt101 maintains genome stability ..................... 4 1.3.1 Cells lacking Rtt101Mms22 are sensitive to DNA damage in S phase ........... 6 1.3.2 Rtt101Mms22 channels repair into an HR-dependent pathway ...................... 7 1.3.3 Rtt101 participates in replication-coupled nucleosome assembly and ubiquitylates histone H3 and FACT ..................................................................... 8 1.3.4 Rtt101 degrades Mms22 in response to DNA damage and controls Rad51 loading ............................................................................................................... 11 1.3.5 Further proposed roles for Rtt101 ............................................................ 11 1.3.6 Conservation of Rtt101Mms22 in human cells ............................................. 12 1.3.7 CUL4 regulates nucleotide excision repair upon UV damage .................. 14 1.3.8 CUL4 regulates cell cycle progression and replication in S phase and in the presence of DNA damage ................................................................................. 15 1.3.9 CUL4 targets histones and affects chromatin state .................................. 16 1.3.10 CUL4 in human disease and cancer....................................................... 17 1.4 Replication fork stalling and restart ................................................................. 18 1.4.1 Causes of replication fork stalling ............................................................. 19 1.4.2 Checkpoint activation ............................................................................... 21 1.4.3 DNA damage tolerance and post-replicative repair .................................. 23 1.4.4 Replication fork reversal ........................................................................... 26 1.4.5 HR-dependent repair of broken replication forks ...................................... 27 1.5 Genomic ribonucleotides and their repair ........................................................ 32 I List of Publications 1.5.1 Direct consequences of misincorporated genomic ribonucleotides .......... 33 1.5.2 Repair of misincorporated genomic ribonucleotides ................................. 34 1.5.3 Impact of misincorporated ribonucleotides on genome stability ............... 38 1.5.4 Implications on human health and disease ............................................... 39 1.6 Scope of this thesis ......................................................................................... 40 2. Results ................................................................................................................. 43 2.1 Rtt101 is required to survive MMS-induced DNA damage .............................. 43 2.1.1 Loss of Mrc1 suppresses MMS sensitivity of cells lacking a functional Rtt101 E3 ubiquitin ligase ............................................................................................. 43 2.1.2 Mrc1 protein levels are not regulated by Rtt101 upon MMS-induced damage .......................................................................................................................... 44 2.1.3 Rtt101 counteracts a replicative function of Mrc1 to promote survival in the presence of MMS .............................................................................................. 46 2.1.4 The checkpoint recovery defect of rtt101∆ cells is alleviated by MRC1 deletion .............................................................................................................. 48 2.1.5 Rtt101 promotes recombination-mediated fork restart by counteracting Mrc1 .......................................................................................................................... 50 2.1.6 Genetic uncoupling of the CMG from DNA synthesis relieves MMS sensitivity of rtt101Δ cells ................................................................................................... 52 2.2 Rtt101 is required to survive accumulating genomic ribonucleotides .............. 54 2.2.1 Loss of Rtt101 is toxic for cells that accumulate genomic ribonucleotides 54 2.2.2 Rtt101 is dispensable for efficient removal of genomic ribonucleotides .... 59 2.2.3 Rtt101 is not required to deal with Top1-mediated damage ..................... 61 2.2.4 RAD51-dependent HR is not epistatic with RTT101 and MRC1 deletion suppresses toxicity caused by accumulated genomic rNMPs ........................... 62 2.2.5 RNase H2 is required post-replicatively in the absence of Rtt101 ............ 65 2.2.6 H3K56Ac is required when genomic ribonucleotides accumulate ............ 68 2.2.7 Nucleosome deposition in RER-defective cells is not altered ................... 70 List of Publications 2.2.8 SILAC-based approach to identify relevant targets of Rtt101 in RER- defective cells .................................................................................................... 75 3. Appendix ............................................................................................................. 79 4. Discussion .......................................................................................................... 87 4.1 The role of Rtt101 at replication forks stalled by MMS-induced DNA lesions .. 87 4.1.1 Rtt101 counteracts a replicative function of Mrc1 ..................................... 88 4.1.2 Modulation of Mrc1 allows restoration of HR ............................................ 88 4.2 Unrepaired genomic ribonucleotides induce replication stress ....................... 91 4.2.1 Rtt101 becomes crucial in the absence of RNase H2 .............................. 92 4.2.2 HR is vital when rNMPs accumulate and MRC1 deletion reduces toxicity 92 4.2.3 RER might be cell cycle regulated ............................................................ 93 4.3 Mass spectrometric approach to identify Rtt101-dependent ubiquitylation when genomic rNMPs accumulate ................................................................................. 94 4.3.1 Dpb2 is an important structural component of the replisome .................... 96 4.3.2 Possible consequences of Rtt101-mediated ubiquitylation of Dpb2 ......... 97 4.4 H3K56Ac and H3 ubiquitylation .................................................................... 100 4.4.1 H3 ubiquitylation becomes crucial when genomic ribonucleotides accumulate ...................................................................................................... 101 4.4.2 Newly synthesized H3-H4 might recruit DNA repair factors ................... 103 4.4.3 The role of Rtt101 in nucleosome assembly and fork stability might be interdependent ................................................................................................ 104 4.5 Rtt101-dependent lesions might have common features .............................. 106 4.5.1 MMS-induced lesions and rNMPs .......................................................... 106 4.5.2 Other types of DNA damage .................................................................. 108 4.6 Implications on human disease: DNA replication defects underlie symptoms of AGS patients ....................................................................................................... 110 4.7 Future perspectives ....................................................................................... 111 5. Materials and Methods ..................................................................................... 113 5.1 Materials ....................................................................................................... 113 III List of Publications 5.1.1 Yeast strains ........................................................................................... 113 5.1.2 Plasmids ................................................................................................. 117 5.1.3 Oligonucleotides ..................................................................................... 118 5.1.4 Liquid media ........................................................................................... 119 5.1.5 Agar plates ............................................................................................. 120 5.1.6 Buffers and solutions .............................................................................. 121 5.1.7 Antibodies ............................................................................................... 122 5.1.8 Enzymes, reagents and commercially available