U N I V E R S I T Y O F C O P E N H A G E N F A C U L T Y O F S C I E N C E PhD Thesis Xu Feng Identification of the archaeal regulatory network in DNA damage response Supervisor: Qunxin She Data of Submission: 12 June 2018 Name of department: Department of Biology Author(s): Xu Feng Title and subtitle: Identification of the archaeal regulatory network in DNA damage response Topic description: The role of a paralog of TFB/TFIIB protein and an orthologue of Orc1/Cdc6 protein during DNA damage-induced transcriptional responses in Sulfolobus islandicus Rey15A was investigated. Supervisor: Qunxin She Submitted on: 12 June 2018 This thesis has been submitted to the PhD School of the Faculty of Science, University of Copenhagen The cover image is an integrated model of the regulatory network of DNA damage response in Sulfolobus. PREFACE The thesis entitled ‘Identification of the archaeal regulatory network in DNA damage response’ was submitted to the Faculty of Science, University of Copenhagen. The work presented in the thesis was carried on at the Danish Archaea Center (DAC), Department of Biology, University of Copenhagen, Denmark, under the supervision of Dr. Qunxin She and with financial support from Danish Council for Independent Research (DFF-4181- 00274) and China Scholarship Council (CSC). The thesis starts with a general overview of mechanisms employed by three domains of life to deal with DNA damage, including DNA damage repair and tolerance pathways. It is followed by an introduction of the current knowledge about the DNA damage response (DDR) across the tree of life. Subsequently, the results obtained during my PhD study are summarized. At last, the thesis goes to its end with the discussion of the results and perspectives for future research. Two papers are included at the end of the thesis, with the first one addressing the function of TFB3 and the second focusing on the functional study of Orc1-2 during DDR regulation in Sulfolobus islandicus Rey15A. These papers are given below: Xu Feng, Mengmeng Sun, Wenyuan Han, Yun Xiang Liang, Qunxin She; A transcriptional factor B paralog functions as an activator to DNA damage-responsive expression in archaea, Nucleic Acids Research, gky236, https://doi.org/10.1093/nar/gky236 Mengmeng Sun, Xu Feng, Zhenzhen Liu, Wenyuan Han, Yun Xiang Liang, Qunxin She; An Orc1/Cdc6 ortholog functions as a key regulator in the DNA damage response in Archaea, Nucleic Acids Research, gky487, https://doi.org/10.1093/nar/gky487 I ACKNOWLEDGEMENTS It has been a wonderful time during my stay in Denmark and I would like to say thanks to everybody that has supported me here. Firstly, I would like to express my sincere gratitude to my supervisor Dr. Qunxin She for his support during my PhD study. His guidance has enlightened me in the past years and will be the most valuable thing for my scientific career. I am grateful to all my current and former labmates in the Danish Archaea Centre and the Molecular Biology of Archaea Lab in Wuhan including Yunxiang Liang, Roger Garrett, Xu Peng, Yongmei Hu, Nan Peng, Yuxia Mei, Zhengjun Chen, Ling Deng, Changyi Zhang, Wenyuan Han, Wenfang Peng, Fei He, Daniel Stiefler-Jensen, Mariana Awayez, Thi Ngoc Hien Phan, Soley Gudbergsdottir, Carlos Leon, Laura Alvarez, Dongqing Jiang, Yingjun Li, Min Ren, Wenqing She, Jingzhong Lin, Yan Zhang, Mingxia Feng, Mengmeng Sun, Tong Guo, Anders Lynge Kjeldsen, Yuvaraj Bhoobalan, Anders Fuglsang, Pavlos Papathanasiou, Anne Louise Grøn Jensen, Weijia Zhang, Zhenzhen Liu and Saifu Pan. It has been a wonderful experience working together with them. My sincere thanks also go to Dr. Li Huang and all the lab members in his lab for their support during my stay in Institute of Microbiology, Chinese Academy of Science at Beijing. My special thanks are dedicated to my best friends including Yingwei Feng, Liuquan Feng, Bin Li, Zhengkun Kuang, Jun Wang, Qishan Zhang and Kaisong Huang. Thanks to them for being there through all those tough times in my life. Last, and most importantly, I would like to express my deep gratitude to all my beloved family members, especially my wife (Jianglan Liao) and my son (Muxin Feng) for their unconditional love and support. II TABLE OF CONTENTS Preface ........................................................................................................................ I Acknowledgements ................................................................................................... II Table of contents ...................................................................................................... III Summary ................................................................................................................... V Sammendrag ........................................................................................................... VII Abstracts .................................................................................................................. IX Abbreviations ........................................................................................................... X Objectives ............................................................................................................... XII Introduction ............................................................................................................... 1 Universal strategies for DNA damage removal .................................................................................. 2 Direct reversal of DNA damage ......................................................................................................... 3 Excision of DNA damage ................................................................................................................... 5 Repair of double strand DNA breaks ................................................................................................ 13 DNA damage tolerance by Translesion DNA synthesis ................................................................... 16 DNA damage response in three domains of Life .............................................................................. 19 ATM/ATR mediated DNA damage signaling pathways in Eukarya ................................................ 19 Multilayer regulations of SOS response in Bacteria ......................................................................... 23 Cellular responses towards DNA damage in Archaea ...................................................................... 26 Transcriptional regulation in Archaea .............................................................................................. 30 Summary of the results .......................................................................................... 33 TFB3 functions as a transcriptional activator for DNA transfer pathway ........................................ 33 Orc1-2 functions as a global regulator essential for DDR in Sulfolobus .......................................... 35 Discussions and future perspectives .................................................................... 38 III References .............................................................................................................. 41 IV SUMMARY DNA damage response (DDR) is essential for the maintenance of genome integrity in all three domains of life, and the process is controlled by evolutionarily unrelated factors in Bacteria and in Eukarya. While DDR is primarily mediated by cleavage of the global repressor, LexA in the former, the process in the latter is mainly orchestrated by two evolutionally conserved kinases, ATM/ATR. Strikingly, none of these DDR regulators have a homologue in Archaea. As a result, it remains elusive as to how organisms in Archaea coordinate cellular processes in response to DNA damage signal (s). Nevertheless, investigation of genome expression upon UV light exposure in Sulfolobus species revealed a number of differentially expressed genes including genes encoding a paralogue of TFB protein (TFB3) and an orthologue of Orc1/Cdc6 protein (Orc1-2). Here, we apply a combination of genetic, biochemical, transcriptome and phylogenetic analysis to investigate their possible roles in archaeal DDR using Sulfolobus islandicus REY15A as the model. Firstly, we constructed tfb3 gene deletion mutant and the transcriptome analysis of the resulting mutant (∆tfb3) revealed that TFB3 is essential for the transcriptional activation of a subset of DDR genes. Phenotypic characterization of ∆tfb3 showed that the mutant loses its ability to form cell aggregates upon DNA damage and is moderately sensitive to DNA damage. Interestingly, CHIP-qPCR analysis showed that TFB3 specifically binds to the promoter region of TFB3-dependent genes, suggesting that TFB3 directly modulates the transcriptional process upon DNA damage. Further, mutagenesis of the TFB3 protein and subsequent functional analysis indicated that the N terminal Zn ribbon and C terminal Coiled-Coil motif are essential for its function in the transcriptional activation. Furthermore, the phylogenetic analysis revealed a co-evolution of TFB3 with its target system (Ced, the Crenarchaeal system for exchange of DNA), suggesting that the TFB3- mediated transcriptional regulation may represent a well conserved DDR regulatory circuit for intercellular DNA transfer in Crenarchaeota. Then, we showed that the previously constructed orc1-2 deletion mutant (∆orc1-2) is hypersensitive to NQO treatment and the transcriptome analysis of the mutant revealed that V Orc1-2 is essential for the global