Biophysical studies of stretched conformations of nucleic acids

Research Project, 2016 – 2019

We will study energetic and mechanistic aspects of DNA, in particular its extended conformations in the context of homologous recombination. Despite the vital importance to all biological organisms for survival, genome maintenance and evolution, and more than 30 years of intense research, the mechanism of the recombination reaction is not yet understood at an atomistic level. Recently, we demonstrated, using single-molecule force spectroscopy, the existence of a 51% extended stable conformation of double-stranded (base-paired) GC-rich DNA, at a transition force of 64 pN. It cannot be a coincidence that the same degree of extension is also found in DNA complexes with recombinase proteins, bacterial RecA and human Rad51. We thus propose that this structural distortion of DNA is related to how these proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that extended DNA conformations undergo a “disproportionation” into sets of stacked bases, preferentially triplets, and that these non-homogeneous structures determine the high-fidelity base pair recognition. Interestingly, a triplet is just the length of a gene codon. Using both single-molecule and bulk experiments, as well as theoretical modeling, we shall test this hypothesis, and also address mechanistic aspects on how RecA deals with DNA and the various roles of ionic and hydrophobic interactions, and of ATP/ADP, and ATP hydrolysis, inside the assembled fibrous RecA-DNA complex. Recent results from model experiments with fluorescence-labeled DNA molecules in polymer environments, make us believe that what we call “hydrophobic catalysis” is of crucial importance here: we postulate that hydrophobic moieties in the protein-nucleic acid complex can lower the activation barrier and facilitate the strand-exchange reaction by stabilizing certain structures.