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ATR Protects the Genome Against R Loops Through a MUS81-Triggered Feedback Loop

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ATR Protects the Genome Against R Loops Through a MUS81-Triggered Feedback Loop ATR Protects the Genome against R loops through a MUS81-Triggered Feedback Loop The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Matos, Dominick Amaral. 2020. ATR Protects the Genome against R loops through a MUS81-Triggered Feedback Loop. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365155 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ATR Protects the Genome against R loops through a MUS81-Triggered Feedback Loop A dissertation presented by Dominick A Matos to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachusetts December 2019 @ 2019 Dominick A Matos All rights reserved. Dissertation Advisor: Lee Zou Dominick A Matos ATR Protects the Genome against R loops through a MUS81-Triggered Feedback Loop Abstract R loops are three-stranded transcription intermediates consisting of RNA:DNA hybrids and displaced single-stranded DNA. Although R loop formation is important for a number of cellular processes, they can cause genomic instability if dysregulated. The R loop-induced genomic instability is largely dependent on DNA replication, suggesting that it may arise from the collisions between R loops and DNA replication forks. How cells respond to the collisions between R loops and replication fork is poorly understood. My study reveals that dysregulated R loop formation during DNA replication activates the ATR checkpoint kinase, the master regulator of the replication stress response. The activation of ATR by aberrant R loops requires reversal of DNA replication forks and DNA cleavage by the MUS81 endonuclease. In contrast to the activation of ATR by replication inhibitors, R loop-induced ATR activation is uniquely dependent upon MUS81. Once activated, ATR protects the genome from R loops by suppressing transcription-replication collisions, enforcing a G2/M arrest, and promoting fork recovery. Additionally, ATR prevents excessive cleavage of reversed forks by MUS81, suggesting that MUS81 triggers a feedback loop in which ATR regulates MUS81 activity at replication forks. Overall, these results reveal an ATR-regulated signaling circuitry induced by collisions of R loops and replication forks, and establish ATR as a key sensor and suppressor of R loop-induced genomic instability. iii Table of Contents Abstract ..................................................................................................................................... iii Table of Contents ..................................................................................................................... iv List of Figures and Tables ....................................................................................................... vi List of Useful Abbreviations .................................................................................................. viii Attributions ............................................................................................................................... ix Acknowledgments ...................................................................................................................... x Chapter 1: Introduction ............................................................................................................... 1 R loops ........................................................................................................................................ 2 R loops are a three-stranded structure formed during transcription ........................................ 2 R loops function in many cellular processes ........................................................................... 5 Dysregulated R loop formation causes genomic instability .................................................... 9 Replication is important for genomic instability induced by R loop accumulation .............. 11 Various proteins are involved in modulating R loop levels .................................................. 15 ATR .......................................................................................................................................... 20 ATR is a master regulator of many DDR pathways, including the replication stress response ............................................................................................................................................... 20 ATR pathway activation requires ssDNA and dsDNA:ssDNA junctions ............................. 20 Activated ATR protects the genome through various mechanisms ...................................... 27 MUS81 ...................................................................................................................................... 32 MUS81 is a substrate-specific endonuclease that can cleave various replication and recombination intermediates ................................................................................................. 32 MUS81 is usually important to the replication stress response ............................................. 35 Contributions of my work ...................................................................................................... 37 Chapter 2: R loop accumulation activates ATR through a unique MUS81-mediated pathway ........................................................................................................................................ 39 Introduction ............................................................................................................................. 41 Results ...................................................................................................................................... 43 R loop accrual activates ATR ................................................................................................ 43 R-loop induced ATR activation is dependent on replication ................................................ 49 MUS81 mediates R loop-induced ATR activation ................................................................ 52 Fork reversal is needed for R loop-induced ATR activation ................................................. 59 ssDNA is needed for R loop-induced ATR activation .......................................................... 62 iv R loops activate ATR via a unique signaling circuitry.......................................................... 65 dsDNA:ssDNA junctions are important to ATR activation during R loop accrual .............. 66 Discussion ................................................................................................................................. 68 Chapter 3: ATR protects the genome against R loop-induced genomic instability through various mechanisms .................................................................................................................... 71 Introduction ............................................................................................................................. 73 Results ...................................................................................................................................... 75 ATR prevents R loop-induced genotoxicity .......................................................................... 75 ATR suppresses transcription-replication collisions ............................................................. 78 ATR promotes DNA synthesis and a G2/M arrest in response to R loops ........................... 82 ATR prevents MUS81-induced DNA damage at reversed forks when R loops accumulate 83 Discussion ................................................................................................................................. 88 Chapter 4: Developing an episome system to study the collisions between R loops and DNA replication forks .......................................................................................................................... 90 Introduction ............................................................................................................................. 91 Results ...................................................................................................................................... 93 Discussion ................................................................................................................................. 99 Chapter 5: Discussion ............................................................................................................... 102 R loops activate the ATR-Chk1 pathway in replicating cells ............................................. 103 Fork reversal is an important process to ATR activation .................................................... 106 MUS81 plays a unique role in activating ATR during R loop accumulation ..................... 108 MUS81 cleavage may generate both ATR-activating substrates ........................................ 110 ATR protects the genome against R loop-induced DNA damage ....................................... 112 ATR suppresses transcription-replication collisions ........................................................... 112 ATR
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