Zl55thesispdf.Pdf (4.442Mb)

Zl55thesispdf.Pdf (4.442Mb)

A MOLECULAR MECHANISM ALLOWING TRANSPOSON TN7 TO TARGET ACTIVE DNA REPLICATION A DISSERTATION PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF CORNELL UNIVERSITY IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY ZAOPING LI JANUARY 2012 ©2012 ZAOPING LI A MOLECULAR MECHANISM ALLOWING TRANSPOSON TN7 TO TARGET ACTIVE DNA REPLICATION Zaoping Li, PhD Cornell University 2012 Transposons are jumping genes that are ubiquitous and abundant in all domains of life. They can move between locations that lack homology within a genome. Transposons drive the evolution of genomes through gene inactivation, expression modulation, and genome rearrangement. The bacterial transposon Tn7 and its relatives are widespread in diverse bacteria, likely due to their ability to control the frequency and targeting of transposition. My work presented here focuses on understanding the molecular mechanism of the TnsABC+E pathway of Tn7 transposition that preferentially targets actively transferring mobile DNA. I was able to establish a sensitive in vitro system for this transposition reaction with purified proteins and gapped DNA substrates preloaded with the β-clamp. The transposition profile recapitulates that observed in vivo, indicating that the minimal features recognized by TnsE to target DNA replication are 3' recessed ends found in target DNA and the β-clamp processivity factor (DnaN). I further show that the TnsE-β interaction is largely conserved among Tn7-like elements; however, this interaction is also species specific. In a heterologous expression study, I found that TnsE homologs from Idiomarina loihiensis and Shewanella baltica only promoted transposition when DnaN from the same host was used in the cell. I propose that TnsE may have evolved to interact with the more variable portion of the clamp to avoid interfering with the normal traffic on the clamp. In an effort to screen for host proteins that may affect TnsE-mediated transposition, I found and confirmed an interaction between TnsE and SeqA, a protein involved in replication initiation control and organizing newly replicated chromosome DNA. Results from genetic studies support a model where TnsE interacts with SeqA to disrupt the SeqA superstructure that tracks with the replication fork. I also show that in wild type background, TnsE is able to direct transposition into the origin region and DNA undergoing leading-strand replication, consistent with the emerging picture that both the leading-strand and lagging-strand DNA replication are essentially discontinuous. These data point to a perspective of using Tn7 as a genetic tool in understanding the replication and repair processes in the cell. BIOGRAPHICAL SKETCH Zaoping Li was born in a small village located in Hunan Province, China. After primary schools, he left his family for a special class in a middle school almost thirty miles away form his hometown and since then he moved further and further on his long road of study. Zaoping went to the School of Public Health at the West China Medical Center of Sichuan University for an undergraduate study and obtained a Bachelor of Medical Science degree. During this five-year period, he was particularly fascinated about Microbiology and Molecular Biology and decided to continue his Master’s study at the same school with Professor Hengchuan Liu. He then transiently worked in Beijing Centers for Disease Control and Prevention as a researcher, but soon realized his real interest was still in science. In 2005 Zaoping had the great opportunity to continue his study for a PhD degree at Cornell University in the United States. After experiencing different aspects of studies in the field of Microbiology by rotation, he joined the laboratory of Dr. Joseph Peters to study transposon Tn7, where he continued his interest in mobile genetic elements and acquired solid training in Genomics and Biochemistry. iii THIS THESIS IS DEDICATED TO MY WIFE, LICHUAN YE, AND MY PARENTS, YUXIAO LI AND SANNIANG LIU iv ACKNOWLEDGEMENTS I would like to acknowledge people who have supported and encouraged me during my years on the academic path. My parents, Yuxiao Li and Sanniang Liu, have been cheerful, encouraging, and supportive for my efforts, but I understand what they have been through to support a higher education with all that they have had. Professor Hengchuan Liu at West China Medical Center of Sichuan Univeristy was the first scientist who extensively inspired my curiosity about science and led me into scientific research from college, and has always been optimistic about my career. I am grateful to the committee members of my PhD study, Dr. Joseph Peters, Dr. Jeffrey Roberts, and Dr. John Helmann, for their supports and insightful advice. I appreciate the guidance from my major advisor Dr. Joseph Peters, who gave me the chance to explore, but also kept my adventures on the right track. Dr. Joseph Peters has also been very helpful in my understanding the new culture. I am thankful to my friends and colleagues in the Microbiology program, especially Adam Parks and Qiaojuan Shi in the Peters’ lab for the wonderful discussions and suggestions. Finally, I am deeply grateful to my wife, Lichuan Ye, who backed me up ever since my Master’s study and has accompanied my pursuit this far. v TABLE OF CONTENTS Biographical Sketch ............................................................................................................ iii Dedication ........................................................................................................................... iv Acknowledgements ............................................................................................................. v Table of Contents ................................................................................................................ vi List of Figures .................................................................................................................... xii List of Tables ..................................................................................................................... xv 1. Introduction ................................................................................................................... 1 1.1. Tn7 transposition functions ....................................................................................... 5 1.1.1. The Tn7L and Tn7R ends are structurally and functionally different ............... 5 1.1.2. The core TnsABC machinery ............................................................................ 8 1.1.2.1. TnsAB the transposase ............................................................................... 9 1.1.2.2. TnsA: a restriction enzyme in recombination ............................... 10 1.1.2.3. TnsB is a member of the large transposes/retroviral integrase superfamily ..................................................................................................... 11 1.1.2.4. TnsC, an AAA+ regulator ......................................................................... 15 1.2. Target selection in the TnsABC+D transposition pathway ................................... 23 1.2.1. Sequence requirements of attTn7 ................................................................. 24 1.2.2. Target DNA binding by TnsD ..................................................................... 25 1.2.3.TnsD interaction with TnsC ......................................................................... 28 vi 1.2.4. Host factors in TnsD-mediated transposition .............................................. 29 1.3. Target selection in the TnsABC+E transposition pathway .................................... 29 1.3.1. TnsE-mediated transposition targets lagging-strand DNA replication ....... 30 1.3.2. Factors important for TnsE targeting of lagging-strand DNA replication ... 31 1.3.3. Other DNA replication processes targeted by TnsE .................................... 39 1.3.4. Why target lagging-strand DNA replication? .............................................. 40 1.4. Recognition of a positive target signal by TnsC .................................................... 43 1.5. Target immunity ..................................................................................................... 45 1.6. Expansion of the Tn7 family by virtue of vertical and horizontal transposition ... 49 1.7. Internal networking of the core machinery ............................................................ 53 1.7.1. Interaction between TnsA and TnsB ........................................................... 53 1.7.2. Interaction between TnsA and TnsC ........................................................... 53 1.7.3. Interaction between TnsB and TnsC ........................................................... 54 1.8. Assembly of the transpososome for TnsABC+D transposition ............................. 60 1.8.1 Pre-transposition complex ............................................................................ 60 1.8.2 The Post-transposition complex ................................................................... 61 1.9. Tn7 as a genetic tool ............................................................................................... 66 1.10. Concluding remarks ............................................................................................. 68 1.11. Acknowledgements .............................................................................................. 69 1.12. References ............................................................................................................

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