Molecular Mechanism, Dynamics, and Energetics of Protein-Mediated Dinucleotide Flipping in a Mismatched DNA: A Computational Study of the RAD4-DNA Complex.

DNA damage alters genetic information and adversely affects gene expression pathways leading to various complex genetic disorders and cancers. DNA repair proteins recognize and rectify DNA damage and mismatches with high fidelity. A critical molecular event that occurs during most protein-mediated DNA repair processes is the extrusion of orphaned bases at the damaged site facilitated by specific repairing enzymes. The molecular-level understanding of the mechanism, dynamics, and energetics of base extrusion is necessary to elucidate the molecular basis of protein-mediated DNA damage repair. The present article investigates the molecular mechanism of dinucleotide extrusion in a mismatched DNA (containing a stretch of three contiguous thymidine-thymidine base pairs) facilitated by Radiation sensitive 4 (RAD4), a key DNA repair protein, on an atom-by-atom basis using molecular dynamics (MD) and umbrella-sampling (US) simulations. Using atomistic models of RAD4-free and RAD4-bound mismatched DNA, the free energy profiles associated with extrusion of mismatched partner bases are determined for both systems. The mismatched bases adopted the most stable intrahelical conformation, and their extrusion was unfavorable in RAD4-free mismatched DNA due to the presence of prohibitively high barriers (>12.0 kcal/mol) along the extrusion pathways. Upon binding of RAD4 to the DNA, the global free energy minimum is shifted to the extrahelical state indicating the key role of RAD4-DNA interactions in catalyzing the dinucleotide base extrusion in the DNA-RAD4 complex. The critical residues of RAD4 contributing to the conformational stability of the mismatched bases are identified, and the energetics of insertion of a β-hairpin of RAD4 into the DNA duplex is examined. The conformational energy landscape-based mechanistic insight into RAD4-mediated base extrusion provided here may serve as a useful baseline to understand the molecular basis of xeroderma pigmentosum C (XPC)-mediated DNA damage repair in humans.