Single DNA studies of architectural proteins involved in bacterial pathogenesis and meiosis in saccharomyces cerevisiae

SINGLE-DNA STUDIES OF ARCHITECTURAL PROTEINS INVOLVED IN BACTERIAL PATHOGENESIS AND MEIOSIS IN SACCHAROMYCES CEREVISIAE LI YOU (M.Sc., UT) A THESIS SUBMITTED FOR THE DEGREE OF Ph.D. OF PHYSICS DEPARTMENT OF PHYSICS NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. —————————— Li You April 2014 i ACKNOWLEDGEMENT Over the last four years, I have benefitted greatly from my interactions with many people in the National University of Singapore. Their professional assistance and coaching are invaluable in my research development. My deepest gratitude goes to my supervisor, A/P YAN Jie. Through my years of study in his group, Yan Jie’s ongoing patience and inspiration have been indispensable in my scientific growth. As a principle investigator, his curiosity and fearlessness have been a huge encouragement in developing my approaches of work and thoughts of experiments. As an advisor, he serves as a model that I admire and respect fully. I am also grateful to our collaborators Prof. Linda Kenney, Prof. K. Muniyappa and Dr. Gauthier. Our collaborations have improved my research in many ways and will continue to be fruitful for years to come. Throughout my life, my parents’ love, encouragement, and understanding have been of utmost importance, and I thank them for everything they did and they are doing. My friends in the lab have provided me with lots of support and useful discussion. I am thankful to have them around. I would like to express my special thanks to Dr. Qu Yuanyuan for being such a good friend always cheering me up. Last but not least, I am thankful to my husband, Thomas Masters, for his love, support, encouragement, critique, and wisdom. As a scientist, he serves as a model of integrity and discipline that has shaped my life philosophy. He is my most trusted friend/mentor and I can never finish my Ph.D degree without him. ii TABLE OF CONTENTS DECLARATION i! ACKNOWLEDGEMENT ii! TABLE OF CONTENTS iii! SUMMARY v! LIST OF FIGURES vi! LIST OF ABBREVIATIONS ix! CHAPTER 1: Introduction .1! 1.1 DNA structure and its coil size 1! 1.2 Overview of chromosomal DNA organization in eukaryotic and prokaryotic cells 8! 1.3 Gene expression and gene silencing 10! 1.4 DNA binding modes of nucleoid-associated proteins (NAPs) and their regulatory functions in bacteria .12! 1.5 Gene-silencing by H-NS and anti-silencing by antagonizing proteins 14! 1.7 Salmonella pathogenesis and the H-NS anti-silencing protein SsrB .21! 1.8 Pathogenic Gram-positive bacterial and current understanding of their nucleiod structuring proteins — Mtb protein MDP1 and mIHF 24! 1.9 Chromosome synapsis during meiosis 26! 1.10 Objectives of this study 30! 1.11 Organization of this thesis .31! CHAPTER 2: Methods and materials .33! 2.1 Single molecule manipulation .33! 2.2 Magnetic tweezers and its application to DNA measurements 34! 2.2.1 Magnetic tweezers setup .34! 2.2.2 Coverslip functionalization .37! 2.2.3 Force Calibration 39! 2.2.4 Single-DNA determination .42! iii 2.2.5 Effects of DNA-binding proteins on FE curves 42! 2.3 Atomic force microscopy .45! 2.3.1 Components of AFM 46! 2.3.2 Principle of AFM 48! 2.3.3 Mica surface modification 52! CHAPTER Single DNA study of the H-NS antagonizing Salmonella enterica response regulator SsrB 55! 3.1 Introduction 55! 3.2 Method 56! 3.3 Results .57! 3.4 Discussion 64! CHAPTER Single DNA study of Mtb protein MDP1 and mIHF 67! 4.1 Introduction 67! 4.2 Method 68! 4.3 Results .72! 4.4 Discussion 85! CHAPTER Single DNA study of Hop1-DNA interaction .89! 5.1 Introduction 89! 5.2 Method 91! 5.3 Results .91! 5.4 Discussion 95! CHAPTER Conclusions .97! BIBLIOGRAPHY 101! LIST OF PUBLICATIONS 116! iv SUMMARY Both eukaryotic and prokaryotic cells must keep their chromosomal DNA well organized. Packaging of millimeter-long DNA molecules inside bacterial cells and centimeter-to-meter-long ones inside eukaryotic cells is achieved through a number of DNA binding architectural proteins. In bacteria, chromosomal DNA is packaged into a tightly folded nucleoid structure by about a dozen nucleoid-associated proteins (NAPs). In eukaryotic cells, DNA is organized into chromatin by histone proteins. Besides packaging DNA, architectural proteins also play other roles in various critical cellular processes, such as gene transcription regulation and cell division. In the preparation of this thesis, I investigated the gene regulation functions of H-NS, a major NAP in Gram-negative bacteria, which controls pathogenesis of Salmonella, Escherichia coli (E.coli) and Yersinia. My studies revealed the mechanism by which H-NS mediated gene-silencing can be relieved through interaction with another protein, SsrB. I also investigated the DNA-binding properties of MDP1 and mIHF, two acid-fast Gram-positive bacteria proteins expressed in Mycobacterium tuberculosis. These proteins are known to control bacterial growth and regulate entry into the dormant state, but the molecular mechanisms were poorly understood. I found that both of these proteins condense DNA into a stable structure. This suggests they function to protect DNA against reactive oxygen intermediate by host immune system and thus play a role in bacterial growth regulation. Finally, I studied the DNA-binding behavior of the protein Hop1, which plays a critical role in aligning two sister chromatids during meiosis in the eukaryote Saccharomyces cerevisiae. I found that Hop1 mediates tight DNA bridging in a zinc ion dependent manner, which has important physiological implications. All these studies were based on direct measurement using a combination of single-DNA manipulation and atomic force imaging technologies to address fundamental questions concerning the mechanical aspects of interactions between architectural proteins and single-DNA molecules. v LIST OF FIGURES Figure 1.1 The basic structure of DNA 2! Figure 1.2 The worm-like chain .4! Figure 1.3 Force-extension curves for DNA 6! Figure 1.4 The common and different features of prokaryotic and eukaryotic cells .8! Figure 1.5 Chromatin and condensed chromosome structure 9! Figure 1.6 The meiotic cell cycle .10! Figure 1.7 The central dogma of molecular biology 11! Figure 1.8 Binding modes of various Gram-negative bacteria NAPs .13! Figure 1.9 Solution structures of H-NS. 16! Figure 1.10 Two H-NS DNA-binding modes 17! Figure 1.11 Possible H-NS gene-silencing mechanisms. 19! Figure 1.12 Illustration of the mechanisms by which anti-silencing proteins antagonize H-NS silencing system 20! Figure 1.13 The TTSS of S. typhimurium 21! Figure 1.14 The structure of SsrBC .22! Figure 1.15 Three-dimensional model of SsrBC bound to DNA. .23! Figure 1.16 Dimerization formation of MDP1 N terminal domain .25! Figure 1.17 CD analysis of mIHF-80 reveals its high content of alpha-helices. .26! Figure 1.18 Illustration of prophase I stages in meiosis I 27! Figure 1.19 Structure of the synaptonemal complex. 28! Figure 2.1 Glass channel for magnetic tweezers experiment. .35! Figure 2.2 Illustration of a DNA tether 36! Figure 2.3 Magnetic tweezers setup .37! Figure 2.4 Steps in the glass coverslip functionalization protocol. .38! Figure 2.5 The inverted pendulum representation of the bead-DNA configuration 40! Figure 2.6 Force-extension response of a double-stranded DNA 43! vi Figure 2.7 Schematic models for protein introduced changes on double-stranded DNA .44! Figure 2.8 Effect of protein nonspecifically binding DNA. 45! Figure 2.9 AFM setup. .47! Figure 2.10 Cantilever deflection detection by optical lever .48! Figure 2.11 The AFM probe tip is typically immersed in the contamination layer above the sample layer .49! Figure 2.12 Qualitative illustration of interaction force versus surface-to-tip distance. 50! Figure 2.13 Two AFM imaging modes are divided by their tip working regions. 51! Figure 2.14 Three imaging modes in vibrating mode 52! Figure 2.15 Schematic illustration of APTES and Glutaraldehyde modified mica surfaces. .53! Figure 3.1 H-NS exhibits distinct behaviors to bind to DNA in buffer with and without magnesium 56! Figure 3.2 Time course for the SsrB folding events. .58! Figure 3.3 FE curves for the SsrB folding events 59! Figure 3.4 SsrB induces strong folding of DNA 60! Figure 3.5 Salt concentration affects the DNA-folding ability of SsrB .61! Figure 3.6 SsrB competes with H-NS in stiffening buffer .62! Figure 3.7 SsrB dose not displace H-NS from DNA in the H-NS DNA-bridging mode. 64! Figure 3.8 Illustration of the mechanism of SsrB 66! Figure 4.1 Finding unfolding steps 71! Figure 4.2. Representative AFM images of MDP1 nucleoprotein in 50 mM KCl 73! Figure 4.3 Representative AFM images of MDP1 nucleoprotein in 200 mM KCl .75! Figure 4.4 DNA compaction by different concentrations of MDP1 77! Figure 4.5 Fitting for the unfolding events using the step-finding algorithm 79! Figure 4.6 Histogram of the probability density against step sizes .80! Figure 4.7 Model of two modes of DNA organization by MDP1. 81! vii Figure 4.8 AFM images of mIHF nucleoprotein complex in 50 mM KCl 83! Figure 4.9 DNA compaction by different concentrations of mIHF in 50 mM KCl. .84! Figure 5.1 S.cerevisiae Hop1 bridges DNA .90! Figure 5.2 Hop1 protein promotes DNA folding .93! Figure 5.3 Hop1 C-terminal domain (Hop1ctd) has much less DNA folding effect .94! viii BIBLIOGRAPHY !"# $%&'()*#+#,$-#.'/0*#,-#')#1%"#"#23'#4)(56)5('#178#9576)0:7#:;#:7)'# A1(%:# >')3:8"# ,# $J# 4)1)# $**:6"# !VQVFQQTGQRUDXXWIQ!"#M5&>'8#M>Z'63170*J# ;:(# A@6%0K1)0:7# :;# 43:()# Z"# A3(:J:*:J'*# 178# A3(:J1)07"# # 23'# A'%%D# $# >:%'65%1(# $LL(:163"#G#'8D#40715'(#$**:601)'*F#GHHH"# !H"# *):('# >P\I%"# A3(:J1)07# 178# A3(:J:*:J'*"# Z7D# A'%%# +0:%:?@# 178# >06(:*6:L@#4)(56)5('#178#9576)0:7#:;#A'%%#e#f0(5*'*#bZ7)'(7')c"#$O10%1&%'#;(:JD# 3))LDSSJ06(:"J1?7')";*5"'85S6'%%*S756%'5*S63(:J1)07"3)J%"# !!"# >1(*):7#$.-#$J:7#$"#>'0:*0*D#A'%%I6@6%'#6:7)(:%*#*35;;%'#178#8'1%"#=1)#N'O# >:%# A'%%# +0:"# GHHQ# Z1(C:#,9"#f1(01)0:7#:;#)3'#;:%807?#178# 8@71J06*# :;# )3'# P*63'(06301# 6:%0# 63(:J:*:J'# /0)3# ?(:/)3# 6:780)0:7*"# >:%# >06(:&0:%"#GH!G"# !Q"# $K1J# 2$-# Z*3031J1# $"# 2/'%O'# *L'60'*# :;# )3'# 756%':08I1**:601)'8# L(:)'07# ;(:J#P*63'(06301#6:%0#I#4'g5'76'#('6:?70)0:7#*L'60;060)@#178#'8# 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:;# L1)3:?'7*# 07*08'# J16(:L31?'*"# >:%# +0:%# A'%%"# GHHa# ,17F!RT!UDQV]IW!H"# M5&>'8# M>ZZ'8# M>ZM$A2Z\=#$=1('*61# 2,-# 4C:C:# @6:&16)'(05J#*J'?J1)0*"#>:%#E'7#E'7')"#!VV]FGaHDQRW#I#V"#M5&>'8# M>ZZZZ1(#!RF!HaT!!UDQQ!QI ]"#M5&>'8#M>Z'63170*)06# 07*0?3)*# 07):# 1# 7:O'%# '_L:()'(I0JL:()'(# *@*)'J# :;# >@6:&16)'(05J# )5&'(65%:*0*# 57(1O'%# 0)*# (:%'# 07# )(1;;06C07?# :;# 0(:7"# M%:*# \7'"# GHH]FXTWUD'GH]R"# M5&>'8# M>Z@6:&16)'(05J# )5&'(65%:*0*"# A'%%# ^:*)# >06(:&'"# GH!X# >1@# !WF!XTWUDaHGI!G"#M5&>'8#M>Z1(F!]TXUD!QGQIXW"#M5&>'8# M>ZAZ-#\*181I\C1#>-#41):3#P9-#`0)18:C:(:#`-#=0*305630#B-#=0C0#>-#')# 1%"# $# ^0*):7'I.0C'# M(:)'07# :;# >@6:&16)'(01# M:**'**'*# 9'((0)07# 45L'(;1J0%@# M(:)'07I.0C'#$6)0O0)@#178#M(:)'6)*#1?107*)#[...]... imaging visualized H-NS /DNA interaction and they found H-NS can fold DNA into hairpin structures (52) Based on this finding, H-NS was considered as a DNA- bridging protein However, in a singleDNA stretching experiment in 2003, another group found H-NS could not fold DNA, instead, it made DNA more rigid (53) These contradicting results were reconciled by a later finding that H-NS in fact has two distinct... causing DNA condensation in the presence of a physiological concentration of magnesium (21) and HU can cause DNA stiffening (20) Fis bends and mediates DNA looping (22, 23) H12 NS family proteins (H-NS and StpA in E coli) bind to DNA cooperatively, leading to formation of rigid nucleoprotein filaments H-NS may also mediate DNA bridging and higher order structures depending on buffer conditions, in particular... through DNA binding remains unclear To address this, in one of my Ph.D research projects, DNA binding properties of a protein called Hop1 were studied to explore its key role in mediating chromosome pairing in Saccharomyces cerevisiae (yeast) that will be discussed in Chapter 5 DNA packaging in prokaryotic cells is different from that in eukaryotic cells because prokaryotes do not have defined nuclei... illustration of TTSS penetration through host cell membrane and injection of bacterial protein (Images from (68) Illustration from (69)) 21 SsrB is one of the critical H-NS anti-silencing proteins (Figure 1.12) in salmonella that positively regulates the expression of diverse virulence genes The Nterminus of SsrB contains the site of phosphorylation and the C-terminus consists of a DNA- binding domain Dimerization... this hypothesis was investigated for the first time using the single- DNA stretching technique 23 1.8 Pathogenic Gram-positive bacterial and current understanding of their nucleiod structuring proteins — Mtb protein MDP1 and mIHF Mycobacterial disease is a major health problem both in developed and developing countries Mtb causes millions of death every year (75) It infects one third of the human population,... genes One of the well-known genes, ORF Rv2986c (hupBMtb) produces a protein that belongs to the histone like family and referred to as mycobacterial DNA binding protein (MDP1) or histone like protein (HLPMt) MDP1 (21.3 kDa) is an NAP constituting 7–10% of the total protein in Mtb (78) It consists of 205 amino acid residues and contains large amounts of alanine (23.78% of total amino acids) and lysine (18.93%),... may prevent DNA being accessed by other proteins including RNAP, thereby causing gene silencing Alternatively, a continuous protein filament on DNA suggests the possibility of it functioning as a physical barrier to block translocation of RNAP along DNA (27) These two potential gene-silencing mechanisms based on the stiffening-binding mode of H-NS are depicted in Figure 1.11 a-b We note that in the physiological... anti-silencing proteins (Figure 1.12) (56) The activities of the anti-silencing proteins play crucial roles in activating pathogenic genes in Salmonella, E.coli and Yersinia, while their anti-silencing mechanisms are 19 poorly understood Essentially, there are two possible ways for anti-silencing proteins to counteract H-NS: compete with H-NS protein to bind to DNA, and compete with DNA to bind to H-NS... level of many genes are suppressed through gene silencing, which is of great importance as proteins are critically involved in the proper function and structure of cells Figure 1.7 The central dogma of molecular biology There are two steps for producing a corresponding protein: transcription and translation Transcription occurs in the nucleus of the cell resulting in the formation of a copy of the information... importance in pathogenesis, it is crucial to understand how it antagonizes H-NS mediated gene silencing One of the main purposes of this thesis is to provide new insights into the mechanism that SsrB employs to counteract H-NS mediated gene silencing Because SsrB is a DNA binding protein, my working hypothesis is that it may destabilize HNS DNA- binding through direct competition with H-NS for DNA binding In . SINGLE- DNA STUDIES OF ARCHITECTURAL PROTEINS INVOLVED IN BACTERIAL PATHOGENESIS AND MEIOSIS IN SACCHAROMYCES CEREVISIAE LI YOU (M.Sc., UT). nucleoid-associated proteins (NAPs). In eukaryotic cells, DNA is organized into chromatin by histone proteins. Besides packaging DNA, architectural proteins also play other roles in various critical. Calibration 39! 2.2.4 Single- DNA determination 42! iv 2.2.5 Effects of DNA- binding proteins on FE curves 42! 2.3 Atomic force microscopy 45! 2.3.1 Components of AFM 46! 2.3.2 Principle of AFM 48! 2.3.3