UC Irvine

The dinB homolog (Dbh) from the thermophilic archaeon Sulfolobus acidocaldarius is a member of the Y-family of translesion DNA polymerases, which are specialized to accurately replicate DNA across from a wide variety of lesions in living cells. Dbh is also a remarkably thermostable polymerase, functioning well at 80°C, the optimum growth temperature of S. acidocaldarius. Herein I present the study of the dynamics of apo Dbh at atomic resolution by hydrogen-deuterium exchange (HDX) NMR, NMR spin relaxation, and molecular dynamics (MD) simulations at two temperatures. In order to interpret the NMR data, it was necessary to assign the backbone resonances of Dbh. To this end, I have assigned the 15N, 1H, and 13C backbone resonance signals at two temperatures (35°C and 50°C) for 86% of the residues of Dbh, which have been published (Moro and Cocco, 2015).

The experiments presented herein demonstrate the remarkable stability of the palm and little finger (LF) domains of Dbh, which remain rigid and well-folded at 50°C. For instance, residues in the palm and LF have protection factors greater than 108 and 109. MD simulations indicate that the LF domain is free to rotate about the linker region with respect to the polymerase core. The LF domain reorientation is much faster at 50°C and can explain the stark difference in Dbh activity at low versus high temperatures. It is also possible that the reorientation of the LF allows the binding of DNA distorted by various types of lesions.

In addition, I performed biophysical experiments (circular dichroism, differential scanning calorimetry, and HDX-NMR) to investigate the possibility of cold denaturation above zero °C for Dbh. Cold denaturation is a well-established phenomenon, but there are few examples of proteins that denature above zero °C. I obtained conflicting evidence for cold denaturation, with CD and DSC indicating some structural change, but no evidence for structural change by HDX-NMR. No evidence was found for a full unfolding of the polypeptide chain of Dbh. Further investigation is merited in order to fully characterize structural changes in Dbh at low temperature.