DNA recognition mechanisms

Research Project, 2021 – 2023

Despite the importance of homologous recombination in cancer, sterility, and CRISPR-Cas contexts, and many years’ intense research, the mechanisms how human Rad51 and bacterial RecA perform search for homology and execute strand exchange are not yet understood at an atomic level: many questions, including why dsDNA is strongly elongated, remain enigmatic. With CRISPR-Cas, after the double-strand break of dsDNA, a bottle-neck is the subsequent insertion of the correct oligonucleotide requiring the cell’s native recombination repair system. Two recent findings could here be clues to mechanistic understanding: ‘hydrophobic catalysis’ and that GC-rich DNA exposed to mechanical pulling force displays a distinct conformation almost exactly 50 % longer than normal DNA, neither phenomenon associated with significant base-pair opening (denaturation) and both involving modulation of stacking energy. We propose both are important for homologous recombination and repair reactions, catalyzing the processes and also improving sequence recognition fidelity. We plan systematic studies of 1) how DNA is affected directly or indirectly by presence of hydrophobic environment, and 2) how structure and efficiency of RecA-DNA and Rad51-DNA complexes depend on hydrophobicity of the L2-loop in recombinase mutants. Solving the recombination enigma, with generalizable implicat­ions to DNA handling in Rad51-CRISPR-Cas9 and other contexts, will pave way for important future medicinal applications.