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Sap1 Dependent Replication Fork Barriers Guide Integration of Ltr Retrotransposons in S

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Sap1 Dependent Replication Fork Barriers Guide Integration of Ltr Retrotransposons in S SAP1 DEPENDENT REPLICATION FORK BARRIERS GUIDE INTEGRATION OF LTR RETROTRANSPOSONS IN S. POMBE By! JAKE ZACHARY JACOBS A Dissertation submitted to the! Graduate School-New Brunswick! Rutgers, The State University of New Jersey !In partial fulfillment of the requirements! For the degree of !Doctor of Philosophy !Graduate Program in Biochemistry !Written under the direction of Dr. Mikel Zaratiegui, Ph.D.! And approved by _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________! New Brunswick, New Jersey! October 2016 ABSTRACT OF THE DISSERTATION SAP1 DEPENDENT REPLICATION FORK BARRIERS GUIDE INTEGRATION OF LTR RETROTRANSPOSONS IN S. POMBE By! JAKE ZACHARY JACOBS Dissertation Director: Dr. Mikel Zaratiegui Long Terminal Repeat retrotransposable elements (LTR-TEs) are a large group of eukaryotic Transposable Elements characterized by flanking repeats in tandem orientation—the LTRs. The LTRs of these elements contain sequences that recruit proteins involved in their expression, replication, silencing, organization, and stability. A successful transposable element must maximize its reproductive amplification without jeopardizing its host, and several characterized LTR-TEs appear to accomplish this through the selection of integration sites away from protein coding sequences. However, despite their high relatedness, a universal mechanism that explains how these parasitic elements avoid coding sequences has not been established. Through sequencing of de novo integration sites of the LTR-TE Tf1 from the fission yeast Schizosaccharomyces pombe, we found a strong integration preference for locations near the binding site of Sap1. Sap1 has been previously shown to be a DNA-binding protein that controls the directionality of DNA replication by causing polar fork arrest. Sap1 mutations that mildly affect binding but strongly affect fork barrier activity decrease Tf1 retrotransposon efficiency ten-fold, indicating that Sap1 replication fork barrier activity is a stronger predictor of Tf1 integration than DNA binding. Further, synthetic Sap1 binding sites placed near DNA origins are only competent at Tf1 recruitment when placed in blocking orientation. Interestingly, the fork arresting activity of an independent factor provided in ii cis can increase the integration efficiency of a barrier-incompetent Sap1 binding site. Thus, both Sap1 binding and replication fork arrest are necessary for Tf1 integration. Together, these data suggest that Sap1 guides insertion of Tf1 by tethering the intasome and blocking the progression of the replication fork, and that the Tf1 transposon uses features of arrested forks to insert into the host genome. Since fork arrest is detectable in many genomic features that recruit LTR-RT integration, such as type III promoters and heterochromatic sequences, these observations point to a universal mechanism for determination of LTR-TE tropism. The questions surrounding the molecular mechanism of Tf1 transposition led to the examination of the CRISPR/Cas9 system as a tool for tethering Tf1 to stalled forks in vivo. However, the CRISPR/Cas9 toolkit had not been developed for S. pombe. Using a novel processed RNA Pol II promoter and the Hammerhead ribozyme we developed a highly efficient CRISPR/Cas9 expression system, leading to >95% modification efficiencies without selection. iii Dedication To all the scientists who came before me, and the millions of hours of thinking and research they performed. Without them, the questions examined in this thesis would not have even been possible to ask. iv Acknowledgements My thesis would not have been nearly as successful or interesting if it were not for the strong support network that helped me through the work presented here. First, I’d like to thank Dr. Zaratiegui for being an absolutely incredible mentor. His experimental support, daily input, and passion for science radiates throughout his laboratory, creating a wonderful atmosphere for scientific discovery. I also would like to thank the other lab members who were critical in the work in this thesis. Vincent, for teaching me the art of the experiment, the importance of never believing your own results, and the value of persistence. Keith, for stepping up and learning a ton of biochemistry and molecular biology when the time was needed, and for assisting with CRISPR/Cas9 experiments. Susanne, for valuable experimental support during the review process and other great discussions. Jesus, for tremendous experimental support, helpful discussions, and importantly, performing, troubleshooting, and analyzing the 3C experiments (Figure 13). Importantly, I also thank my incredibly supportive family. First, my parents who constantly supported my childhood curiosity, and never let it fade. Also, my wife Julie, who remained supportive through the whole process, and never asked too much of me when I was burdened. Also, my dog Wilbur and newborn baby Jordan, who greeted me with much needed excitement and joy every time I came home. As a final note, it should be noted that the work contained within Chapter II and V are mostly reproduced works from scientific publications completed as part of this thesis (Jacobs, Ciccaglione, Tournier, & Zaratiegui, 2014; Jacobs et al., 2015). v Table Of Contents Title page ......................................................................................................................................................... i Abstract ........................................................................................................................................................... ii Dedication ...................................................................................................................................................... iv Acknowledgements ........................................................................................................................................ v Table of Contents .......................................................................................................................................... vi List of Tables ................................................................................................................................................ vii List of Illustrations ...................................................................................................................................... viii Chapter I - An Introduction! to Transposons and CRISPR/Cas9 .............................................................. 1 1. Transposons ........................................................................................................................................... 1 1-1. The Biological Impact of Transposon Activity .............................................................................. 1 1-2. Transposon Mobilization: From Colonization to Target Site Selection ......................................... 7 1-3. LTR Retrotransposons: Structure and Function ........................................................................... 13 1-4. LTR Retrotransposon Target Site Selection in Schizosaccharomyces pombe .............................. 19 2. CRISPR/Cas9 Genome Editing in S. Pombe .................................................................................... 26 2-1. Genome Editing History and Principles ....................................................................................... 26 2-2. Genome Modification in Fission Yeast ........................................................................................ 34 Chapter II - Arrested Replication Forks Guide Tf1 Integration ............................................................ 38 1. Introduction ......................................................................................................................................... 38 2. Materials and Methods ....................................................................................................................... 40 3. Results .................................................................................................................................................. 59 4. Discussion ............................................................................................................................................. 81 Chapter III – Genetic Examination of the Role of RFB on Tf1 Transposition ...................................... 84 1. Introduction ......................................................................................................................................... 84 2. Materials and Methods ....................................................................................................................... 89 3. Results .................................................................................................................................................. 94 4. Discussion ........................................................................................................................................... 106 Chapter IV - Efficient Retrotransposon Targeting and Periodicity Is Accomplished Through The Tf1 Chromodomain .......................................................................................................................................... 109 1. Introduction ......................................................................................................................................
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