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Defining Sites of Replication Fork Collapse Caused by Atr Inhibition University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Defining Sites Of Replication orkF Collapse Caused By Atr Inhibition Nishita Kalpendu Shastri University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, Molecular Biology Commons, and the Pharmacology Commons Recommended Citation Shastri, Nishita Kalpendu, "Defining Sites Of Replication orkF Collapse Caused By Atr Inhibition" (2017). Publicly Accessible Penn Dissertations. 2581. https://repository.upenn.edu/edissertations/2581 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2581 For more information, please contact [email protected]. Defining Sites Of Replication orkF Collapse Caused By Atr Inhibition Abstract DEFINING SITES OF REPLICATION FORK COLLAPSE CAUSED BY ATR INHIBITION Nishita K. Shastri Eric J. Brown Replication stress, characterized by stalling of DNA replication and the accumulation of abnormal replication intermediates, has been linked to the genomic instability observed in cancer. Previous studies have defined specific genomic sequences that ear difficulto t replicate to be more vulnerable to replication-associated breaks and rearrangements. However, many of these sequences have been identified through indirect and potentially biased approaches. To identify DNA sequences that contribute to replication-associated genomic instability, I will describe genome-wide screens I have performed to determine the location, sequence, and frequency of replication perturbations within the mammalian genome upon replication stress. Ataxia telangiectasia and Rad3-related protein (ATR) is a checkpoint kinase that is a key upstream regulator of the response pathway to replication fork stalling during replication stress that prevents fork collapse. Through inhibition of this response pathway in mouse embryonic fibroblasts, my aims are to 1) characterize regions that lead to frequent replication fork stalling and collapse, and 2) further define genomic regions that become processed into double-strand breaks. Since replication protein A (RPA) binds to single-stranded DNA that becomes exposed when replication forks stall, RPA ChIP-Seq has been performed to map sites of frequently collapsed replication forks; however, not all stalled replication forks result in breaks. To differentiate a replication fork that has simply stalled from a fork that has become sensitized to double-strand break formation, I developed and applied a novel and specific break-detection assay, BrITL. With these complementary approaches to map replication-problematic loci, subsequent bioinformatics methods have been utilized to characterize features of the identified genomic egionsr that make it prone to fork collapse and detrimental DNA break formation when cells experience replication stress. While well-established difficult-to-replicate sequences (e.g. triplet and telomere repeats) exhibited enhanced fork collapse in RPA ChIP’d cells exposed to replication stress, these sequences were overshadowed by sites composed of previously uncharacterized simple tandem repeats. Circular dichroism and thermal difference absorption spectra indicate that the most commonly observed simple repeat at RPA-enriched sites (CAGAGG) folds into a stable intramolecular secondary structure and is sufficiento t stall DNA replication in vitro and in vivo. BrITL analysis confirmed that these epetitivr e regions of RPA accumulation are also sites of DNA breakage. Interestingly, a majority of break sites identified by BrITL do not associate with RPA accumulation, but rather tend to locate around inverted retroelements that are predicted to form highly stable intrastrand stem-loop structures. Due to the lack of available ssDNA at these potential hairpin-forming sites, RPA accumulation would be limited. Overall, my studies represent the first unbiased identification of mammalian genomic sites that are vulnerable to replication stress and rely on ATR for stability. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Eric J. Brown Keywords ATR, DNA damage, DNA replication stress, Fork collapse, RPA Subject Categories Cell Biology | Molecular Biology | Pharmacology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2581 DEFINING SITES OF REPLICATION FORK COLLAPSE CAUSED BY ATR INHIBITION Nishita K. Shastri A DISSERTATION in Pharmacology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation __________________________ Eric J. Brown, PhD Associate Professor of Cancer Biology Graduate Group Chairperson __________________________ Julie A. Blendy, PhD, Professor of Pharmacology Dissertation Committee Klaus Kaestner, PhD, Thomas and Evelyn Suor Butterworth Professor in Genetics (Chair) Gerd Blobel, MD, PhD, Frank E. Weise III Professor of Pediatrics F. Bradley Johnson, MD, PhD, Associate Professor of Pathology and Laboratory Medicine I would like to dedicate this to my family: my brother, Anujit, my sister, Ankita, and my parents, Kalpendu and Bhavna Shastri. I would also like to dedicate this to my grandparents, Biharilal and Jaya Shah, and Ranjit and Anila Shastri. They have always been my greatest support and my highest inspiration. I hope I can make them proud. ii ACKNOWLEDGMENTS I would like to thank the following people for all their help: Yu-Chen Tsai, who started the work with optimization and application of the RPA ChIP-Seq protocol on MEFs, and provided the constructs for the structural assays as well as for the in vivo and in vitro studies on fork pausing. Dillon Maloney, who provided the essential bioinformatics work that led to our customized pipeline for ChIP-Seq and BrITL deep sequencing data analysis. He was also integral in developing the program REQer for our in-depth look at repetitive sequences in our ChIP-Seq data sets. Our bioinformatics collaborators (Jonathan Schug, Rafael Casellas, Marei Dose) who provided key analyses on our sequences and advice on demonstrating the significance of our findings. Our collaborators for their work on demonstrating both the formation of unique structures by our identified sequences (Liliya Yatsunyk, Jessica Chen, Deondre Jordan, Barrett Powell) and the resultant stalling of the polymerase complex in our in vitro and ex vivo systems (Kristin Eckert, Suzanne Hile). The Functional Genomics Core (FGC) at Penn for deep sequencing RPA ChIP- Seq and BrITL samples, and the Institute for Biomedical Informatics (IBI) at Penn for work done to identify inverted repeats in the mouse genome. The Brown lab members for their considerable support and advice, and, most of all, Eric Brown, for his incredible mentorship. iii ABSTRACT DEFINING SITES OF REPLICATION FORK COLLAPSE CAUSED BY ATR INHIBITION Nishita K. Shastri Eric J. Brown Replication stress, characterized by stalling of DNA replication and the accumulation of abnormal replication intermediates, has been linked to the genomic instability observed in cancer. Previous studies have defined specific genomic sequences that are difficult to replicate to be more vulnerable to replication-associated breaks and rearrangements. However, many of these sequences have been identified through indirect and potentially biased approaches. To identify DNA sequences that contribute to replication-associated genomic instability, I will describe genome-wide screens I have performed to determine the location, sequence, and frequency of replication perturbations within the mammalian genome upon replication stress. Ataxia telangiectasia and Rad3-related protein (ATR) is a checkpoint kinase that is a key upstream regulator of the response pathway to replication fork stalling during replication stress that prevents fork collapse. Through inhibition of this response pathway in mouse embryonic fibroblasts, my aims are to 1) characterize regions that lead to frequent replication fork stalling and collapse, and 2) further define genomic regions that become processed into double- strand breaks. Since replication protein A (RPA) binds to single-stranded DNA that becomes exposed when replication forks stall, RPA ChIP-Seq has been performed to map sites of frequently collapsed replication forks; however, not all stalled replication forks result in breaks. To differentiate a replication fork that has simply stalled from a fork iv that has become sensitized to double-strand break formation, I developed and applied a novel and specific break-detection assay, BrITL. With these complementary approaches to map replication-problematic loci, subsequent bioinformatics methods have been utilized to characterize features of the identified genomic regions that make it prone to fork collapse and detrimental DNA break formation when cells experience replication stress. While well-established difficult-to-replicate sequences (e.g. triplet and telomere repeats) exhibited enhanced fork collapse in RPA ChIP’d cells exposed to replication stress, these sequences were overshadowed by sites composed of previously uncharacterized simple tandem repeats. Circular dichroism and thermal difference absorption spectra indicate that the most commonly observed simple repeat at RPA- enriched sites (CAGAGG) folds into a stable intramolecular secondary structure and is sufficient to stall DNA replication in
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