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
