Dynamics of the bypass polymerase, dinB homolog.

Abstract

The dinB homolog (Dbh) polymerase is a member of the Y-family of translesion DNA polymerases, able to synthesize DNA across damaged DNA template. Since Dbh is derived from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning at high temperatures where mesophilic proteins would be denatured. Here we use NMR and Circular Dichroism (CD) to compare protein stability at a wide range of temperatures. 1H-15N HSQC spectra show a change of tertiary structures starting at 25 °C and progression at colder temperatures (15, 5 °C). CD show retentions of secondary structure with a slight decrease percent helicity and an increase in percent beta strand as temperature decrease. We have analyzed hydrogen exchange measurement at 35°C and 50°C to investigate the folding dynamics of Dbh. We observe the moderate modulation of dynamics upon heating. Core residues in the palm and little finger domain, especially those in the interior of the β-sheets and interior face of α-helices, are key in maintaining the stability of Dbh; with the palm providing a stable platform for the active site residues. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature.