DNA polymerase activity in water-structured and confined environment of reverse micelles

DNA polymerases are the key enzymes of DNA replication and repair. These proteins in living cells are functioning not as isolated entities but in multiprotein complexes, and their work are carefully regulated. The main factors determining the enzyme activity are the structure and dynamics of 'biological' water. Reverse micelles (nano-sized water droplets dispersed in a continuous oil phase) are the simple model systems where the structure and dynamics of water are controlled. In this work, the activities and processivity of Klenow fragment of E. coli DNA polymerase I, thermostable Tte DNA polymerase, and HIV-1 reverse transcriptase are investigated as a function of the water pool size. Klenow fragment was more active on poly(rA)-oligo(dT) in reverse micelles compared with the water buffer with activity being increased upon increasing water content. Tte polymerase was more active at low water content. HIV-1 reverse transcriptase revealed comparable activity and processivity on poly(rA)-oligo(dT) in the water buffer and reverse micelles at water content of 15-40%. Thus, the polymerase activity appears in certain range of water concentration and depends on the local polarity determining the protein 'expansion', microviscosity inside nano-droplets determining the enzyme dynamics, and nucleic acid hydration degree.