A University of Sussex PhD thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details Role of replicative primase in lesion bypass during DNA replication A thesis submitted to the University of Sussex for the degree of Doctor of Philosophy Farimah Borazjani Gholami February 2017 I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree. Farimah Borazjani Gholami University of Sussex Farimah. Borazjani Gholami Doctor of Philosophy Biochemistry Role of replicative primase in lesion bypass during DNA replication Summary Maintenance of genome integrity and stability is fundamental for any form of life. This is complicated as DNA is highly reactive and always under attack from a wide range of endogenous and exogenous sources which can lead to different damages in the DNA. To preserve the integrity of DNA replication, cells hav evolved a variety of DNA repair pathways. DNA damage tolerance mechanisms serve as the last line of defence to rescue the stalled replications forks. TLS, error-prone type of DNA damage tolerance, acts to bypass DNA lesions and allows continuation of DNA replication. Surprisingly majority of archaeal species lack canonical TLS polymerases. This poses a question as to how archaea restart stalled replication in the absence of TLS or lesion repair pathways. This thesis establishes that archaeal replicative primase (PriS/L), a member of the archaeo- eukaryotic primase (AEP) superfamily, possessing both primase and polymerase activities, is able to bypass the most common oxidative damages and highly distorting lesions caused by UV radiation. It has been postulated that archaeal replicative polymerases (Pol B and Pol D family Pols) can bind tightly to the deaminated bases uracil and cause replication fork stalling four bases prior to dU. A specific mechanism for resuming replication of uracil containing DNA by PriS/L is suggested in this thesis. In this thesis, we also reported how the enzymatic activities of archaeal PriS/L are regulated. Here, it is demonstrated that in contrast to archaeal replicative polymerases, single-strand binding proteins (RPA) significantly limit the polymerase activity of PriS/L. The remaining results chapter interrogates the possible interactions between PriS/L and RPA. Finally, the attempts to reconstitute an archaeal CMG complex in vitro, with the aim of shedding light on the role of archaeal replicative primase in replication-specific TLS are described. Acknowledgments Firstly, I would like to express my sincere gratitude to my supervisor, Prof. Aidan Doherty, for giving me the opportunity to do my PhD in his laboratory on such an interesting project and for his continued support and guidance in all the time of research. Beside my supervisor, I would like to thank all the current and former members of Doherty laboratory, including, Stanislaw, Laura, Nigel, Ben, Tom, Pierre, Peter, Alfredo, Przemek and Katie for their invaluable scientific expertise and guidance. A special thanks to Stanislaw who taught me so much and also for his insightful comments and suggestions. I would like to thank Laura for helping me and showing me how to function in a lab. I would like to express my special appreciation to my committee members, Dr. Jo Murray, Dr. Jon Baxter and Dr. Antony Oliver for their helpful advice. Thank you to all people of the GDSC, for providing a good atmosphere to work. I would like to thank my recently graduated friends and also Sahar, my late night lab buddy. My deepest thank goes to my mother, father, brother and my aunt and rest of my family. Their patient and support during these past years enabled me to complete this thesis. A special word of thanks also goes to my husband, Masoud Hasrati for his encouragement and continued support. I would like to dedicate this work to my parents Table of Contents Abbreviations ................................................................................................................. i List of Figures .............................................................................................................. iv List of Table ................................................................................................................. vii Chapter 1 ........................................................................................................................ 1 Introduction.................................................................................................................... 1 1.1 DNA ......................................................................................................................... 2 1.1.1. Biological function of DNA ............................................................................... 2 1.1.2. Structure of DNA.............................................................................................. 2 1.2. Genome replication.............................................................................................. 4 1.3. DNA damage ......................................................................................................... 6 1.3.1. Ionizing radiation-induced DNA damage......................................................... 7 1.3.2. UV-induced DNA damage ............................................................................... 7 1.3.3. Oxidative DNA damage ................................................................................... 9 1.3.4. Alkylating agents and their effects on DNA................................................... 11 1.3.5. Abasic sites in DNA ....................................................................................... 12 1.3.6. Cytosine deamination .................................................................................... 12 1.4. DNA repair pathways ......................................................................................... 13 1.4.1. Direct reversal repair ..................................................................................... 15 1.4.2. Excision Repair .............................................................................................. 16 1.4.3. Homologous recombination ........................................................................... 19 1.5. DNA damage tolerance ...................................................................................... 20 1.6. Bacterial DNA replication .................................................................................. 25 1.7. Eukaryotic DNA replication............................................................................... 26 1.8. Archaeal replisomes .......................................................................................... 30 1.8.1. Archaeal replication origin ............................................................................. 31 1.8.2 MCM helicase ................................................................................................. 35 1.8.3 GINS ............................................................................................................... 36 1.8.4. Archaeal Cdc45 ............................................................................................. 39 1.8.5. Single-stranded DNA binding protein ............................................................ 40 1.9. DNA polymerases............................................................................................... 41 1.9.1. Discovery of DNA polymerases..................................................................... 41 1.9.2. Classification of DNA polymerases ............................................................... 42 1.9.3. Structure and function of DNA polymerases ................................................. 48 1.9.4. Fidelity of DNA polymerase ........................................................................... 50 1.9.5. Proofreading mechanisms ............................................................................. 53 1.10. DNA Primase..................................................................................................... 54 1.10.1. DNA primases- evolution and structure ...................................................... 55 1.10.2. Evolutionary history of AEPs ....................................................................... 56 1.10.3. Structural analysis of the AEP superfamily ................................................. 58 1.10.4. Eukaryotic AEP primases ............................................................................ 62 1.10.5. AEPs involve in NHEJ ................................................................................. 64 1.10.6. Viral AEP primases ...................................................................................... 65 1.10.7. Discovery of second eukaryotic AEP, PrimPol ........................................... 67 1.10.8. Archaeal AEP primases............................................................................... 69 Chapter 2 ......................................................................................................................74