Binding Sites of theE. ColiDNA Recombinase Protein to the ssDNA: A Computational Study

Homologous recombination (HR) is a major pathway for the repair of double strand breaks, which is of primary importance for genomic stability and survival of all organisms. The crucial step in HR is the formation of the nucleoprotein filament composed by a single stranded DNA chain surrounded by the recombinases protein. The filament leads the search for an undamaged homologue, a sister-chromatid or homologous chromosome, as a template for the repair process. The theoretical study presented in this work is aimed to increase the understanding of the mechanism of interaction between a trimer of the recombinase enzyme in the Escherichia coli, EcRecA, with a model system of single stranded DNA, dT(9). The molecular dynamics (MD) calculations were performed using the Amber 10 computer code. The binding free energies are calculated with the Molecular Mechanics (MM) Poisson-Boltzmann (PB) and Generalized Born (GB) solvent accessible surface area (SA), MM-PB(GB)SA model. The study is extended by the use of the Principal Component Analysis (PCA) to reduce the dimensionality of the conformational space. Several mutants are considered and the corresponding calculated binding free energies are compared. An experimental correlation between the binding free energy and the enzyme activity is absent in the literature, however our results confirm how the wild type RecA has a higher binding affinity compared to the mutants examined. Moreover, for all cases considered, a significant contribution of the binding free energy is due to one amino-acid, R196, located in the binding loop 2 of the enzyme. This is consistent with the complete loss of the enzyme activity for any mutation on that point.