A Thesis Entitled Naturally-Occurring Fusion Between the Regulatory And
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A Thesis entitled Naturally-Occurring Fusion Between the Regulatory and Catalytic Components of Type IIP Restriction-Modification Systems by Jixiao Liang Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biomedical Sciences _________________________________________ Dr. Robert Blumenthal, Committee Chair _________________________________________ Dr. Steve Patrick, Committee Member _________________________________________ Dr. Jason Huntley, Committee Member _________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo December 2013 Copyright 2013, Jixiao Liang This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Naturally-Occurring Fusion Between the Regulatory and Catalytic Components of Type IIP Restriction-Modification Systems by Jixiao Liang Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biomedical Sciences The University of Toledo December 2013 Restriction-modification (R-M) systems play key roles in controlling gene flow among bacteria and archaea, and their own genetic mobility depends critically on their regulation, but the regulation of these systems is poorly understood. The PvuII R-M system is a Type IIP R-M system in that the protective DNA methyltransferase (MTase) is a separate and independently-active protein from the potentially lethal restriction endonuclease (REase). PvuII is one of the best studied of the R-M systems that use a positive feedback regulatory loop, involving a transcriptional regulator called C protein, to delay expression of the REase relative to that of the MTase. This allows protective methylation of a new host cell’s DNA before the REase is produced. In searching for R- M systems related to PvuII, in order to study evolution and variation of its regulatory system, a putative system was found in the genome sequence of the bacterium Niabella soli strain DSM 19437, in which the regulatory C protein and the REase are translationally fused. The hypothesis is that N. soli truly produces a fused C-R protein, and that it is active as both a REase and as an autogenous regulator. The genes for the N. soli R-M system were synthesized, produced and purified with affinity tags, and the iii production of full-length C-REase fusion protein was confirmed. The dual activity of the fusion protein was determined by in vitro restriction of known DNAs, and in vivo transcriptional activation of a lacZ fusion to the promoter on which the C protein acts. iv This work is dedicated to my parents, Zhao-jun Liang and Gui-ying Xu for their love and support. Acknowledgements This thesis and the associated research would not have been possible without the ever-patient guidance of my mentor, Dr. Robert Blumenthal. I would like to express my sincere gratitude to my major advisor Dr. Robert Blumenthal for his continuous support of my graduate study and research, for his patience, encouragement, guidance and support. He recognizes my strength and weakness, which keep me motivated. I am also grateful for all his advice about life, career and everything else. I would additionally like to thank my committee members, Dr. Jason Huntley and Dr. Steve Patrick for their valuable time, constructive suggestions, and criticisms during my study. Further, for her constant support as an instructor in lab and a friend in life, I would like to sincerely thank my lab mate Dr. Kristen Williams. Also, my friends Dr. Guo-ping Ren and Dr. Gang Ren have offered me valuable advice and help on my experiments. Last but not least, I would like to thank all the students, faculty, and staff in the Medical Microbiology and Immunology Department. Thank you all! vi Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ............................................................................................................ vi Table of Contents .............................................................................................................. vii List of Figures .................................................................................................................. viii Chapter 1: Literature Review ...............................................................................................1 Chapter 2: Materials and Methods .....................................................................................13 Chapter 3: Results………………………………………………………………………..22 Chapter 4: Discussion and Conclusion ..............................................................................33 References ..........................................................................................................................39 vii List of Figures Figure1 Complex formed by R.PvuII and its cognate DNA. Figure2 PvuII R-M system control region. Figure3 Structure of C. AhdI. Figure4 Sequence of synthesized NsoJS138I R-M system. Figure5 Vector map of constructed plasmids Figure6 Alignment of CR fusion proteins orthologous to C.PvuII and R.PvuII. Figure7 Test of CR fusion protein production. Figure8 Test of CR fusion protein production. Figure9 Assessment of REase activity in CR.NsoJS138I. Figure10 Confirmation of specific digestion conditions. Figure11 Assessment of C activity in CR.NsoJS138I. Figure12 Possible interactions of C-REase fusion polypeptides. viii Chapter 1 Literature Review 1. Restriction-modification (R-M) systems The biological phenomenon of restriction and modification were first recognized in the early 1950s, and the first R-M system was cloned in E. coli in the late 1970s [1]. R- M systems are present in the great majority of bacteria and archaea, with more than 3000 being found to date (most by detecting MTase gene sequences) [2]. As the term indicates, a typical R-M system comprises two activities: a restriction endonuclease (REase) that cleaves DNA at a target sequence, and a methyltransferase (MTase) that modifies the same sequence to protect it from the cognate REase [2]. Four broad types of R-M systems have been reported so far, each with unique characteristics, and the two enzymes have been combined into a single multi-subunit protein in some of the systems [3]. However in Type IIP R-M systems, the REase and MTase separately execute their opposing intracellular enzymatic activities [3]. 1.1 Restriction Endonuclease (REase) The REase catalyzes the cleavage of double-stranded DNA, generally on both strands. REases recognize specific sequences on the target DNA, and the cleavage occurs 1 via hydrolysis of one phosphate-deoxyribose bond in the backbone of each DNA strand [4]. Typically, such enzymatic activity takes place without energy input, but commonly requires Mg2+ or a similar divalent cation; some REases also require or are stimulated by, ATP or S-adenosylmethionine (AdoMet) [5]. REases appear to come from very different backgrounds, and are difficult to identify from their sequences alone [6-8]. 1.2 Modification Methyltransferase (MTase) REase cleavage of DNA could be lethal to cells producing R-M systems. To protect endogenous DNA from REase, the paired (cognate) MTase catalyzes addition of a methyl group to one nucleotide in each strand of the recognition sequence, with the identities and positions varying from MTase to MTase [9]. AdoMet always serves as the methyl donor and is thus an essential cofactor for methylation [10]. The sensitivity of the REase of R-M systems to methylation on the recognition sequences usually prevents cleavage of endogenous DNA. However, while cleavage can be prevented by the cognate methylation, noncognate methylation occurring elsewhere in the recognition sequence may or may not prevent the cleavage [11]. 1.3 Types of restriction modification systems R-M systems are classified based on enzyme composition and cofactor requirements, recognition sequence symmetry, and cleavage position [3, 12]. Because my research defines a new subtype of R-M system, in which the REase and regulatory C protein are fused, it is appropriate to describe the various known types of R-M systems. 2 1.3.1 Type I Systems Type I systems are considered as the most complex R-M systems, as they consist of three polypeptides: R (Restriction), M (Modification) and S (Specificity). These form a complex that can both cleave and methylate DNA in an energy (ATP) dependent manner, and about half of the bacterial genomes contain closely linked-genes that are predicted to code for these three polypeptides, based on screening of the present database of complete genomic sequences [13]. Furthermore, the fact that cleavage occurs at a considerable distance away from the recognition site in most cases, makes it difficult to visualize the discrete bands by gel electrophoresis [14]. So these enzymes have substantial biological significance, but have not yet found major biotechnological uses. 1.3.2 Type II Systems Type II systems are believed to be the simplest and most prevalent R-M systems. As opposed to type I systems, Type II REase and MTase act independently without the need of a specificity protein, and each has its own simple catalytic requirement: REase requires Mg2+ (or similar divalent cation) and MTase requires AdoMet [14]. Type IIP REases are generally active after they dimerize and form homodimers, while