Dynamics and Mechanism of Dna-Bending Proteins in Binding Site Recognition

ISBN-13: 9783319832180 / Angielski / Miękka / 2018 / 199 str.

Yogambigai Velmurugu

Spis treści

I INTRODUCTION……………………………….. 1

1.1 Protein-DNA interactions …………… 1

1.2 Sequence-dependent DNA deformability and its role in target recognition 3

1.2.1 Free energy cost for local deformation of DNA. …………… 6

1.2.2 Sequence-dependent base-pair opening rate measured by NMR imino proton exchange …………… 8

1.2.3 How do site-specific proteins search for their target sites on genomic DNA? …… 9

1.2.4 How do site-specific proteins recognize their target sites? …………… 11

1.2.5 Conformational capture or protein-induced DNA bending…………… 14

1.2.6 Measurements of DNA binding and bending kinetics …………… 14

1.2.7 Competition between 1-D diffusion and binding-site recognition: the “speed-stability” paradox. ……………… 16

1.3 Experimental techniques to study dynamics of protein-DNA interactions… 17

1.3.1 Laser temperature-jump spectroscopy. ……… 18

1.4 Thesis Overview…………… 20

II METHODS………………………………………………………….. 33

2.1 Equilibrium measurements…………… 33

2.2 Laser Temperature Jump technique…………… 33

2.2.1 Laser Temperature jump spectrometer ……… 35

2.2.2 Theoretical estimation of the size of the T-jump…………… 38

2.2.3 Photo-acoustic effects and cavitation. …………… 39

2.2.4 Estimation of temperature jump using reference sample in a T-jump experiment…………… 40

2.2.5 T-jump recovery kinetics…………… 43

2.2.6 Discrete single- or double-exponential decay convoluted with T-jump recovery 46

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2.2.7 Acquisition and matching of relaxation traces measured over different time-scales 46

2.2.8 Maximum entropy analysis…………… 48

2.3 Equilibrium FRET measurements…………… 50

2.4 Nucleotide analog 2-Aminopurine (2AP) …………… 59

2.5 Fraction of Protein and DNA in complex at Equilibrium…………… 61

III Integration Host Factor (IHF)-DNA interaction………………………….67

3.1 Introduction…………… 67

3.1.1 Integration host factor (IHF) …………… 67

3.1.2 IHF binds to the minor groove on DNA and recognizes its specific site via indirect readout…………… 68

3.1.3 Structure of IHF-H’ complex…………… 69

3.1.4 Background of IHF/H’ interaction dynamics …………… 73

3.1.5 Binding site recognition versus protein diffusional search…………… 78

3.2 Results…………… 80

3.2.1 DNA bending kinetics in the IHF – H’ complex are biphasic…………… 80

3.2.2 The slow phase occurs on the same time scale as spontaneous bp

opening at a kink site. …………… 82

3.2.3 Introducing mismatches at the site of the kinks affects the slow

phase but not the fast phase. …………… 84

3.2.4 DNA bending rates in the slow phase of IHF- TT8AT

complex reflect enhanced base-pair opening rates in mismatched DNA…………… 90

3.2.5 DNA modifications away from the kink sites have no effect

on either of the two rates. …………… 92

3.2.6 Two plausible scenarios for biphasic relaxation kinetics…………… 95

3.2.7 Salt-dependence of the fast and slow components. …………… 95

3.2.8 Protein mutations distal to the kink sites affect affinity and bending rate of slow phase…………… 101

3.2.9 Control experiments to rule out contributions to the relaxation kinetics from dye dynamics or dye interactions with protein or DNA……………. 108

3.3 Discussion…………… 112

3.4 Concluding Remarks…………… 117

IV LESION RECOGNITION BY XERODERMA PIGMENTOSUM C (XPC)

PROTEIN………………………………………………………...124

4.1 Introduction…………… 124

4.1.1 Nucleotide excision repair (NER) …………… 124

4.1.2 Experimental design…………… 132

4.2 Method…………… 135

4.2.1 Preparation of double-stranded DNA substrates. …………… 135

4.2.2 Preparation of Rad4–Rad23 complexes. …………… 135

4.2.3 Duplex melting temperatures of mismatched and

undamaged/matched DNA. ………….…………… 139

4.2.4 Apparent binding affinities (Kd,app) determined by electrophoretic

mobility shift assays……………………….…………… 137

4.2.5 Equilibrium FRET temperature scan experiments

with tCo/tCnitro probes. ……………. 138

4.2.6 Acquisition and analyses of T-jump relaxation traces. …………… 139

4.3 Results …………… 144

4.3.1 Kinetics of Rad4 (wild type) induced DNA opening rate…………… 144

4.3.2 tCo and tCnitro FRET pair as probes for sensing changes in

DNA helical structure. …………… 159

4.3.3 DNA bending dynamics measured with extrinsically

attached FRET pair/ AN7…………… 189

4.4 Discussion………………………… 196

4.4.1 Rad4/XPC induced nucleotide flipping/ Open dynamics measured with 2AP

probe……………………………… 196

4.4.2 Rad4/XPC induced helical distortion dynamics measured using tco/tcniro…………… 198

4.4.3 Rad4/XPC induced DNA bending dynamics measured using

TAMRA/Cy5 FRET pair…………… 204

4.5 Conclusion…………… 205

V DNA MISMATCH REPAIR……………………………………………… 213

5.1 Introduction…………… 213

5.1.1 Structural Studies on MutS bound to mismatched DNA…………… 216

5.1.2 What role does the intrinsic flexibility of DNA play in the

mismatch recognition and subsequent repair? …………… 218

5.1.3 Dynamics of DNA binding and bending by MutS. …………… 219

5.2 Results…………… 220

5.2.1 Taq MutS binding to mismatch (T-bulge) DNA as probed by 2AP…………… 221

5.2.2 Taq MutS binding to mismatch (T-bulge) DNA as probed by FRET pair………… 227

5.2.3 MutS binding to mismatch (T-bulge) DNA as

probed by 2AP (in DNA) and Trp (in MutS) …………… 233

5.3 Discussion…………… 238

5.4 Conclusion…………… 240

O autorze

Yogambigai Velmurugu was awarded the PhD degree by the University of Illinois, Chicago, in 2015. 

Blurb

Using a novel approach that combines high temporal resolution of the laser T-jump technique with unique sets of fluorescent probes, this study unveils previously unresolved DNA dynamics during search and recognition by an architectural DNA bending protein and two DNA damage recognition proteins.

Many cellular processes involve special proteins that bind to specific DNA sites with high affinity. How these proteins recognize their sites while rapidly searching amidst ~3 billion nonspecific sites in genomic DNA remains an outstanding puzzle. Structural studies show that proteins severely deform DNA at specific sites and indicate that DNA deformability is a key factor in site-specific recognition. However, the dynamics of DNA deformations have been difficult to capture, thus obscuring our understanding of recognition mechanisms. 

The experiments presented in this thesis uncover, for the first time, rapid (~100-500 microseconds) DNA unwinding/bending attributed to nonspecific interrogation, prior to slower (~5-50 milliseconds) DNA kinking/bending/nucleotide-flipping during recognition. These results help illuminate how a searching protein interrogates DNA deformability and eventually “stumbles” upon its target site. Submillisecond interrogation may promote preferential stalling of the rapidly scanning protein at cognate sites, thus enabling site-recognition. Such multi-step search-interrogation-recognition processes through dynamic conformational changes may well be common to the recognition mechanisms for diverse DNA-binding proteins.