Unveiling Mechanistic Details of Macromolecular Interactions: Structural Design and Molecular Modelling of DNA-Protein Systems in Their Active State
Sammanfattning: Molecular structure is fundamental for understanding mechanisms of molecular interactions. This applies not least to understanding biological function: every biological cell, whether bacterial or human, is an immensely complex system of thousands of molecules that exist in constant motion and interaction with each other. Structural properties of the two main classes of biological macromolecules, nucleic acids and proteins, have been studied in this Thesis focusing on functional interactions of both DNA and the DNA-binding enzyme human recombinase Rad51. DNA is a highly polymorphic molecule and its plasticity may be important for its function. Conformational mechanics of the DNA helix was addressed to understand interactions with dumbbell-shaped ruthenium(II) polypyridyl compounds, known for their remarkable ability to slowly thread one of their bulky centres through a tightly packed DNA stack, probably invoking large transient conformational rearrangements of the helix. Thread-intercalation rate is accelerated by several orders of magnitude if the DNA target sequence is a stretch of at least ten base pairs of AT, as well as by the hydrophobicity of the auxiliary “dumbbell” ligands: counterintuitively, a smaller and less hydrophobic compound takes longer times to thread. It is hypothesized that thread-intercalation might be facilitated by an A-like DNA conformation, induced by the outside binding of the Ru(II) compounds. An NMR study, aiming to solve a thread-intercalated structure of the binuclear Ru(II)-DNA complex, resulted in a groove-binding geometry, probably representing an initial binding mode preceding intercalation, a result emphasizing the elusiveness and immense complexity of the threading process. Turning back to simpler monomeric propeller-shaped Ru(II) compounds it was deduced that, despite acting as classic intercalators, they can “read out” the chirality of the DNA helix by enantiospecifically kinking it, in a fashion analogous to several families of operatory DNA binding proteins. Another operatory protein, human recombinase Rad51, that facilitates homologous recombination, the process of exchanging near identical-sequence DNA strands, is also mechanically acting on DNA, by stretching it. A 3-D high-resolution model structure of human Rad51 filament was solved by a combination of polarized-light spectroscopic data and molecular modelling. Highlighted by the model some interesting structural features could be addressed: strategic locations of two putative DNA binding loops inside the protein filament; as well as location of a putative ATP binding site at the interface between two protein subunits and in direct proximity to a supposed location of DNA – could hint about DNA docking mechanism and potential role of ATP in the protein function. The Rad51-DNA model has proven itself useful also in a follow-up study on the stimulatory role of Ca2+ in the strand exchange reaction by human Rad51. A mechanism is proposed involving a high affinity DNA binding state of the Rad51 filament induced by Ca2+, regulatorily crucial for the search of homology and subsequent DNA strands exchange.
Denna avhandling är EVENTUELLT nedladdningsbar som PDF. Kolla denna länk för att se om den går att ladda ner.