The University of Toledo The University of Toledo Digital Repository Theses and Dissertations 2013 Naturally-occurring fusion between the regulatory and catalytic components of type IIP restrictionmodification systems Jixiao Liang The University of Toledo Follow this and additional works at: http://utdr.utoledo.edu/theses-dissertations Recommended Citation Liang, Jixiao, "Naturally-occurring fusion between the regulatory and catalytic components of type IIP restriction-modification systems" (2013) Theses and Dissertations Paper 134 This Thesis is brought to you for free and open access by The University of Toledo Digital Repository It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of The University of Toledo Digital Repository For more information, please see the repository's About page 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 RM 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 Literature Review 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] RM 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 REase will be formed at certain time with each of them being dimerized, respectively If this is true, the competitive dimerization not only indicates that the two portions could not become active simultaneously, but also might have implications on the relative timing of MTase and REase appearance after the R-M system genes enter a new host cell First, I consider effects on the appearance of REase activity If the C interface is stronger than the R interface, active REase would only appear at later times, after a higher concentration of fused polypeptide had accumulated On the other hand, a stronger REase interface (compared to the C interface) would result in the early appearance of small amounts of REase activity, which might require DNA repair Second, I consider the effects on timing of gene expression A stronger C interface (relative to REase) would result in an earlier and sharper induction threshold in the positive feedback loop; while a stronger REase interface may lead to the longer time for the positive feedback loop to cross the threshold for high expression of the fusion gene, giving more time for protective methylation to occur (even if some DNA damage results from the low level of early REase activity) Either way, this competitive dimerization model seems to provide the most obvious (and testable) hypotheses of the three interaction modes for possible selective advantages of forming C-REase fusions The significance of this work manifests itself not only in identification and characterization of this novel Type II R-M system bearing the C-R fusion, but also provides a perspective of how the structural variation of certain proteins could have affected the functional regulation, thus contributing to the understanding of bacterial evolution 36 Conclusions: R-M systems closely related to PvuII (as judged by similarity of the REase sequences) have diverse regulatory mechanisms Most resemble PvuII in having a separate regulatory (C) protein, and one of these fusion proteins, from the bacterium Niabella soli, is active both as a REase and as a C protein Fusions between C proteins and REases have not previously been characterized These results reinforce the evidence for modularity among RM system proteins, and raise important questions about the possible selective advantages of C-REase fusion, including implications of these fusions 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