Dithioated Phosphates in DNA Duplex Thermodynamics and Protein-DNA Interactions


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Phosphorodithioated DNA contains sulfur atoms at both non-bridging phosphoryl oxygen positions. As an achiral chemical modification of the internucleotide linkage it imparts altered biophysical properties to the phosphate moiety, but still retains normal DNA characteristics such as the ability to form duplexes with complementary strands and to be involved in protein-DNA interactions. Sparse information is currently known about the biophysical effects of phosphorodithioation in DNA, and this dissertation addresses dithioated phosphates in three fundamental aspects of DNA research: DNA synthesis, DNA duplex hybridization thermodynamics, and protein-DNA interactions. The hybridization of two complementary DNA strands into helical duplexes is a well-studied biophysical phenomenon. Normal DNA duplex thermodynamics are predicted from its sequence using unified nearest-neighbor (NN) base pair doublet parameter sets, but currently there is a lack of information for phosphorodithioate modifications. Here a study of 40 different 11-bp duplexes containing all dithioate NNs is presented. Fitting with a single phosphorodithioate parameter revealed the general effect for dithioation: the duplex melting temperature is lowered, the transition free energy becomes less favorable (∆∆G° = +0.67 kcal/mol), the enthalpic term becomes less favorable (∆∆H° = +3.1 iv kcal/mol), and the entropic term becomes more favorable (∆∆S° = +7.7 cal/mol·K). Significant variation from these values was apparent and depended on sequence context, and further modeling revealed particular importance of the purine/pyrimidine identity of base 3' to the dithioate. Numerous phosphorodithioate difference-value parameters were modeled and the sets presented here are suitable for predicting thermodynamic values of short dithioated DNA duplexes from unified normal DNA NN parameter values. Protein-DNA interactions are mediated primarily through ion pair contacts between side-chains and the phosphate backbone. The effect of site-specific phosphorodithioation at the contact point with a lysine NH3+ group was investigated for the HoxD9 homeodomain system using biophysical experiments and nuclear magnetic resonance (NMR). Binding experiments revealed a nearly three-fold increase in duplex affinity upon dithioation of a single phosphate, but isothermal titration calorimetry could not detect an enthalpic difference that would contribute to this apparent lower free energy term. NMR 15N relaxation and hydrogen-bond scalar 15N−31P J-couplings (h3JNP) were then used to investigate the dynamics of the intermolecular ion pairs that form between a lysine NH3+ groups and the DNA phosphate or phosphorodithioate backbone. Surprisingly, order parameters and correlation times for C−N bond rotation and reorientation of the lysine NH3+ groups indicated that in general, NH3+ groups involved in intermolecular ion pairs at the protein−DNA interface are highly dynamic. There is a transition between contact ion pair (CIP) and separated ion pair (SIP) states that occurs on the sub-nanosecond time scale, which should lower the entropic costs of protein-DNA association. Phosphorodithioation was shown to increase the dynamics of the associating lysine NH3+ group, and the overall increase in entropy (i.e. reorientational + rotational) upon dithioation of the ion pair was estimated to be ~ +0.8 cal/mol·K more favorable. Together with binding data, these suggest that the affinity enhancement observed after phosphorodithioation at the location of Lys57 in HoxD9 is due primarily to entropic enhancement.



Phosphorodithioate, dithioated DNA, sulfur-for-oxygen substitution, nucleic acid thermodynamics, DNA Nearest Neighbor, ion pair, CIP, SIP, NMR, ITC