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Scholarly Works (3 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2015)
© 2015 Springer Science+Business Media Dordrecht The dinB homolog (Dbh) 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. Lesioned bases block the progression of high-fidelity polymerases and cause detrimental replication fork stalling; Y-family polymerases can bypass these lesions. The active site of the translesion synthesis polymerase is more open than that of a replicative polymerase; consequently Dbh polymerizes with low fidelity. Bypass polymerases also have low processivity. Short extension past the lesion allows the high-fidelity polymerase to switch back onto the site of replication. Dbh and the other Y-family polymerases have been used as structural models to investigate the mechanisms of DNA polymerization and lesion bypass. Many high-resolution crystal structures of Y-family polymerases have been reported. NMR dynamics studies can complement these structures by providing a measure of protein motions. Here we report the 15N, 1H, and 13C backbone resonance assignments at two temperatures (35 and 50 °C) for Sulfolobus acidocaldarius Dbh polymerase. Backbone resonance assignments have been obtained for 86 % of the residues. The polymerase active site is assigned as well as the majority of residues in each of the four domains.
    Cover page: 1H, 13C, and 15N backbone resonance assignments of the full-length 40 kDa S. acidocaldarius Y-family DNA polymerase, dinB homolog
    • Thesis
    • Peer Reviewed
    UC Irvine Electronic Theses and Dissertations (2015)

    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.

      Cover page: Characterization of the Dynamics and Thermostability of Y-family translesion DNA polymerase DbhCreative Commons 'BY-NC-ND' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2024)
      The DinB homolog polymerase (Dbh) is a member of the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning over a wide range of temperatures. Existing X-ray structures were determined at temperatures where the protein is least active. Here we use NMR and circular dichroism to understand how the structure and dynamics of Dbh are affected by temperature (2°C-65°C) and metal ion binding in solution. We measured hydrogen exchange protection factors, temperature coefficients, and chemical shift perturbations with and without magnesium and manganese. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature. Hydrogen exchange protection factors and temperature coefficients reveal that both the thumb and finger domains are very dynamic relative to the palm and little-finger (LF) domains. These trends remain true at high temperature with dynamics increasing as temperatures increase from 35°C to 50°C. Notably, NMR spectra show that the Dbh tertiary structure cold denatures beginning at 25°C and increases in denaturation as the temperature is lowered to 5°C with little change observed by CD. Above 35°C, chemical shift perturbation analysis in the presence and absence of magnesium and manganese reveals three ion binding sites, without DNA bound. In contrast, these bound metals are not apparent in any Dbh crystal structures of the protein without DNA. Two ion binding sites are confirmed to be near the active site, as reported in other Y-family polymerases, and we report a novel ion binding site in the LF domain. Thus, the solution-state structure of the Dbh polymerase is distinct from that of the solid-state structures and shows an unusually high cold denaturation temperature.
        Cover page: Dynamics and thermal stability of the bypass polymerase, DinB homolog (Dbh).Creative Commons 'BY' version 4.0 license
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