The DNA Flexibility&Interaction Between RNA And PA N-Terminal Domain Of The Avian Influenza Polymerase

Double-stranded DNA is among the stiffest biopolymers. whose conformational flexibility is related to many vital biological processes. Under physiological conditions, the electrostatic repulsion contributes greatly to the DNA rigidity along with the strong base-stacking interaction. Until recently, people still do not have a consensus on which effect contributes most to DNA stiffness and the effect of metal ions in the solution on DNA flexibility.In most of studies on DNA flexibility, the bases are usually treated as planar and the effect of its conformational flexibility on DNA structure is not been considered. In the present work, we studied the conformational flexibility of bases in paired state in the gas phase at room temperature using Car-Parrinello molecular dynamics (MD) approach. The pairing influence on the stiffness of rings is analyzed based on the molec-ular structure of nucleobases and constraints caused by pairing. We prove that the flex-ibilities of pyrimidine rings in isolated state have subtle correlation with the degree of aromaticity of the rings. The pairings in nucleic base pairs induce the rings to be more rigid for guanine (G), thymine (T), and uracil (U) but more flexible for adenine (A) and the same for cytosine (C).Considering thermodynamics effects, we used ab initio constrained MD and meta-dynamics to investigate the mechanism of proton transfer in guanine-cytosine (GC) and adenine-thymien (AT) base pairs in gas phase. It is shown that double proton transfer (DPT) in the GC base pair is a concerted and asynchronous mechanism, and three path-ways with a similar free energy barrier start from the canonical GC and end up in its "rare" imino-enol tautomer. DPT in AT base pair is a stepwise and an asynchronous mechanism. Although a lot of paper has been published on this topic, we present new and more detailed informations for DPT in GC and GC by constructing the full dimen-sional free energy surface for the reactions using fancy methods. The third topic is about the electrostatic interaction effect in DNA backbone on its conformational flexibility by comparing the structural properties of DNA at "neu-tralized" and normal states. We find that the "neutralization" affects little on the major groove of d(GC)10d(CG)10but induces that of d(AT)10d(TA)10to be narrower in the NaCl electrolyte because the former duplex has a stronger interaction with Na-than the later one thus it feels less effects from the "neutralization". In the MgCl2electrolyte, the major groove widths of both types of DNA chains become wider after "neutralization" because of the strong interaction between Mg2-ions with DNA. For the minor groove, all become narrower after "neutralization" in two types of electrolytes. Additionally, charge reduction in the backbone phosphate induces d(AT)10·d(AT)10to bend more se-vere than d(GC)10·d(CG)i0compared to their normal states. And principle component analysis (PCA) shows that d(GC)10·d(CG)10is more rigid, whereas d(AT)10·d(TA)10is more flexibile in their neutralized states. As for helical parameters, calculations in-dicate that the base pairs become stiffer due to the "neutralization". This phenomena also indicate that electrostatic interaction in the DNA backbone influences the structural properties of stacking base pairs which are buried deeply in DNA.The last chapter of the present thesis is about the interactions between RNA and PA N-terminal (PAN) domain of the avian influenza polymerase. Based on the crystal structure of PAN, we construct one-and two-Mg2+PAN-RNA complexes using MD simulations. We carefully check the validity through long time MD simulations. The stable and reasonable active sites of constructed complexes further prove our specula-tions. Our simulations validate the binding of a second metal ion in the presence of the RNA strand, and therefore support a two-metal ions catalysis mechanism in which K134works as the catalytic lysine. Nevertheless, at suboptimal magnesium concentra-tions, an alternative mechanism is also possible, with K137as the catalytic lysine and H41as the general base.