Fate of Transcription Elongation Complexes Stalled by DNA Damage and Elongation Inhibitors by Rachel Krasich Department of Biochemistry Duke University Date:_______________________ Approved: ___________________________ Kenneth N. Kreuzer, Supervisor ___________________________ Arno L. Greenleaf ___________________________ Meta J. Kuehn ___________________________ Christopher Nicchitta ___________________________ Pei Zhou Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University 2014 i v Abstract Fate of Transcription Elongation Complexes Stalled by DNA Damage and Elongation Inhibitors by Rachel Krasich Department of Biochemistry Duke University Date:_______________________ Approved: ___________________________ Kenneth N. Kreuzer, Supervisor ___________________________ Arno L. Greenleaf ___________________________ Meta J. Kuehn ___________________________ Christopher Nicchitta ___________________________ Pei Zhou An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University 2014 i v Copyright by Rachel Krasich 2014 Abstract Transcription, the essential process by which cells translate genetic information stored in DNA into RNA, is a highly regulated and discontinuous process. Elongation is frequently blocked by DNA damage, pause sites, or intrinsic or external inhibitors. Due to the essential nature of transcription, the cell has numerous ways of dealing with these blockages to transcription, only some of which are understood. We examined the fate of RNA polymerase stalled by DNA-protein crosslinks (DPCs), as well as elongation inhibitors Streptolydigin (Stl) and Actinomycin D (ActD). Our lab previously showed the importance of the tmRNA system for survival during DPC-formation, implying that transcription and translation are blocked by DPCs. Using 5-azacytidine-cytosine methyltransferase crosslinks as a model system for DPCs in E. coli, we tested knockout mutants of factors known to affect transcription using cell growth assays. Of these mutants, only dksA mutants were hypersensitive. However, western blots for tmRNA tagging showed that dksA mutants have increased rather than decreased tmRNA tagging, indicating that another unknown factor is responsible for enabling tmRNA activity. We also used the same cell growth assay to look for potential repair pathways for DPCs and found that dnaK knockouts were slightly resistant to DPCs while dnaJ knockous are sensitive. We propose a potential DnaK-independent role for DnaJ in DPC repair. iv To isolate the effects of transcription elongation stalling, we treated cells with the elongation inhibitors Stl and ActD. Previous in vivo studies implied that Stl-inhibited polymerases are released from the DNA transcript via an unknown release factor. Using cell growth assays, Western blots for tmRNA tagging, and in vitro studies, we showed the transcription-coupled repair (TCR) factor Mfd is responsible for releasing both Stl- and ActD-stalled RNAP. We also treated rpoB mutants with ActD and found several ActD resistant mutants, implying alterations to RNAP are sufficient to eliminate ActD inhibition. The tmRNA western blots also implied that Mfd has termination abilities in wildtype cells, leading us to perform RNAseq analysis on mfd knockout and overexpressing cells. We found that global transcription patterns are changed by altering Mfd levels, thus allowing us to propose a novel transcription regulatory role for Mfd. Our studies show that polymerases stalled by DPCs and by elongation inhibitors are resolved by different mechanisms, emphasizing the importance of understanding the different pathways invovled in transcription elongation clearing. We also show that inhibitors such as ActD are effective against cells overexpressing the TCR pathway, which could have potential implications for the treatment of platinum-resistant tumors that have elevated levels of TCR. v Dedication I would like to dedicate this dissertation to my Grandpa Krasich, who was my true role model in life. vi Contents Abstract ......................................................................................................................................... iv List of Tables .................................................................................................................................. x List of Figures ............................................................................................................................... xi Acknowledgements .................................................................................................................. xiii 1. Background ................................................................................................................................ 1 1.1 Stages of transcription ..................................................................................................... 2 1.2 Physical block to RNAP: DNA-Protein Crosslinks ...................................................... 8 1.2.1. Causes and consequences of DNA-protein crosslinks .......................................... 8 1.2.2. Mechanism of MTase binding to cytidine and 5-azacytidine ............................ 10 1.2.3. Significance of 5-azacytidine study........................................................................ 14 1.3 Exogenous elongation inhibitors .................................................................................. 16 1.3.1 Streptolydigin ............................................................................................................ 16 1.3.2. Actinomycin D .......................................................................................................... 18 1.4 Fate of paused or stalled RNAP ................................................................................... 19 1.5 Major questions to be addressed .................................................................................. 22 2. Functions that protect Escherichia coli from DNA-protein crosslinks .............................. 24 2.1 Introduction ..................................................................................................................... 24 2.2 Results .............................................................................................................................. 25 2.3 Discussion ........................................................................................................................ 53 2.4 Materials and Methods .................................................................................................. 59 vii 3. Examining the mechanisms of elongation inhibition for Actinomycin D and Streptolydigin .............................................................................................................................. 63 3.1 Introduction ..................................................................................................................... 63 3.2 Results .............................................................................................................................. 65 3.3 Discussion ....................................................................................................................... 80 3.4 Materials and Methods .................................................................................................. 84 4. Fate of transcription elongation complexes stalled by exogenous elongation inhibitors ...................................................................................................................................... 89 4.1 Introduction ..................................................................................................................... 89 4.2. Results ............................................................................................................................. 91 4.3 Discussion ...................................................................................................................... 105 4.4 Materials and Methods ................................................................................................ 109 5. Mfd alters global transcription patterns in undamaged Escherichia coli cells ............... 114 5.1 Introduction ................................................................................................................... 114 5.2 Results ............................................................................................................................ 117 5.3 Discussion ...................................................................................................................... 140 5.4 Materials and Methods ................................................................................................ 146 6. Conclusions and Future Directions .................................................................................... 151 6.1 Summary of Results ..................................................................................................... 151 6.2 “Chain-reaction” model for DPC consequences ...................................................... 157 6.3 Repair of DPCs .............................................................................................................. 159 6.4 Actinomycin D mechanism of inhibition .................................................................. 160 6.5 Factors that recognize Stl-stalled RNAP ................................................................... 162 viii 6.6 In vitro elongation studies with Streptolydigin and Actinomycin D .................... 163 6.7 Identifying the targets of Mfd termination in wildtype cells ................................. 165 6.8 Final Remarks ............................................................................................................... 166 Appendix I……………………………………………………………………………………..169 References……………………………………………………………………………………...170 Biography ................................................................................................................................... 188 ix List of Tables Table 1: FIC values from the aza-C / bicyclomycin titrations. .............................................. 34 Table 2: Summary of transposon-insertion mutants hypersensitive to DPC inducer aza-C ....................................................................................................................................................... 42 Table 3: Sequence summary of the dinD::lacZ fusion construct ........................................... 48 Table 4a: RpoB mutations that lead to ActD resistance ......................................................... 77 Table 5: Stl hypersensitivity profile results for transcription elongation and termination factors ............................................................................................................................................ 95 Table 6: Genes overexpressed >2-fold (p>0.05) in mfd cells ................................................. 123 Table 7: Genes repressed >2-fold (p>0.05) in mfd cells ......................................................... 124 Table 8a: GO annotations with significant representation in genes overexpressed >2-fold in mfd cells. ................................................................................................................................. 125 Table 8b: GO annotations with significant representation in genes repressed >2-fold in mfd cells…………………………………………………………………………….128 x