Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Winter 12-15-2019 Activation and Regulation of the ALKBH3-ASCC Alkylation Repair Pathway Josh Brickner Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Biochemistry Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Brickner, Josh, "Activation and Regulation of the ALKBH3-ASCC Alkylation Repair Pathway" (2019). Arts & Sciences Electronic Theses and Dissertations. 1991. https://openscholarship.wustl.edu/art_sci_etds/1991 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Molecular Cell Biology Dissertation Examination Committee: Nima Mosammaparast, Chair Susana Gonzalo Albert Kim Alessandro Vindigni Zhongsheng You Hani Zaher Activation and Regulation of the ALKBH3-ASCC Alkylation Repair Pathway by Joshua Brickner A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2019 St. Louis, Missouri © 2019, Joshua Brickner Table of Contents List of Figures ................................................................................................................................. v Acknowledgments........................................................................................................................ viii Abstract of the Dissertation ........................................................................................................... xi Chapter 1: Introduction ................................................................................................................... 1 1.1 Signaling Cascades in Double Strand Break Repair ........................................................ 1 1.1.1 DSB Recognition and Phosphorylation ................................................................................ 1 1.1.2 Ubiquitination During DSB Repair ....................................................................................... 2 1.2 The Cellular Response to Alkylation Damage ................................................................. 5 1.2.1 Characteristics of DNA Alkylation Adducts ......................................................................... 5 1.2.2 Repair by the O6-methylguanine DNA Methyltransferase ................................................... 6 1.2.3 Direct Reversal by the AlkB Family of Iron Dioxygenases.................................................. 8 1.2.4 The Base Excision Repair (BER) Pathway ........................................................................... 9 1.2.5 Regulation of Alkylation Repair Pathways by Ubiquitin Signaling ................................... 10 1.3 The ALKBH3-ASCC Complex in Alkylation Repair.................................................... 11 1.3.1 The ASCC3 Helicase .......................................................................................................... 11 1.3.2 ASCC1 and ASCC2 ............................................................................................................ 12 1.4 The Interplay Between Transcription and DNA Damage .............................................. 14 1.4.1 DNA Damage Responses and Transcription ....................................................................... 14 1.4.2 Alkylated Lesions and Effects on Transcription ................................................................. 16 1.4.3 ASCC3 and Transcription ................................................................................................... 17 1.4.4 RNA Alkylation and the ASCC-ALKBH3 Repair Pathway ............................................... 18 Chapter 2: A Ubiquitin-Dependent Signaling Axis Specific for ALKBH-Mediated DNA Dealkylation Repair ...................................................................................................................... 21 2.1 Abstract .......................................................................................................................... 21 2.2 Introduction .................................................................................................................... 22 2.3 Results ............................................................................................................................ 23 2.3.1 The ASCC Complex Forms Nuclear Foci During Alkylation Damage .............................. 23 2.3.2 The ASCC Complex Co-localizes with the Spliceosome ................................................... 26 2.3.3 ASCC2 Binds to K63-polyubiquitin via its CUE Domain .................................................. 31 2.3.4 ASCC-ALKBH3 Recruitment is Dependent upon ASCC2 ................................................ 34 ii 2.3.5 ASCC2 is Necessary for Alkylation Damage Resistance ................................................... 39 2.3.6 ASCC2 Recruits ASCC-ALKBH3 Through K63-Ub Recognition .................................... 39 2.3.7 The E3 Ligase RNF113A is Necessary for ASCC2 Foci Formation .................................. 46 2.3.8 BRR2 Ubiquitylation is Critical for ASCC-ALKBH3 Recruitment ................................... 49 2.3.9 RNF113A Mutation is Implicated in X-linked Trichothiodystrophy .................................. 52 2.4 Discussion ...................................................................................................................... 56 2.5 Materials and Methods ................................................................................................... 57 Chapter 3: RNA Ligase-Like Domain in ASCC1 Regulates ASCC Complex Function during Alkylation Damage ....................................................................................................................... 68 3.1 Abstract .......................................................................................................................... 68 3.2 Introduction .................................................................................................................... 69 3.3 Results ............................................................................................................................ 71 3.3.1 ASCC1 Interacts Directly with ASCC3 .............................................................................. 71 3.3.2 ASCC1 Forms Nuclear Foci in the Absence of Alkylation Damage .................................. 74 3.3.3 ASCC1 Modulates Alkylation-Induced ASCC3 Foci Formation ....................................... 76 3.3.4 Deletion Analysis of ASCC1 Reveals Modular Functional Domains ................................ 80 3.3.5 A Putative RNA-Binding Domain in ASCC1 Regulates ASCC Function ......................... 83 3.3.6 ASCC1 is Important for Alkylation Resistance .................................................................. 86 3.4 Discussion ...................................................................................................................... 89 3.5 Materials and Methods ................................................................................................... 92 Chapter 4: Aberrant RNA Methylation Triggers Recruitment of the ASCC-ALKBH3 Repair Complex ........................................................................................................................................ 97 4.1 Abstract .......................................................................................................................... 97 4.2 Introduction .................................................................................................................... 98 4.3 Results .......................................................................................................................... 100 4.3.1 RNA Alkylation is Necessary to Mediate ASCC Recruitment ......................................... 100 4.3.2 RNA Alkylation is Sufficient to Mediate ASCC Recruitment ......................................... 103 4.3.2 ASCC-mediated Transcriptional Repression in Response to Aberrant RNA Alkylation . 106 4.3.3 Selective Activation of RNF113A E3 Ligase Activity Upon Alkylation ......................... 108 4.4 Discussion .................................................................................................................... 111 4.5 Materials and Methods ................................................................................................. 117 Chapter 5: Conclusion and Future Directions ............................................................................. 124 iii 5.1 Ubiquitin Recognition by ASCC2 in Alkylation Repair .............................................. 125 5.1.1 Conclusions ....................................................................................................................... 125 5.1.2 Future Directions............................................................................................................... 128 5.2 ASCC-ALKBH3 Complex Coordination by ASCC1 .................................................. 137 5.2.1 Conclusions ......................................................................................................................