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Conformational changes during nucleotide selection by Sulfolobus solfataricus DNA polymerase Dpo4
Robert L Eoff1, Raymundo Sanchez-Ponce, F Peter Guengerich
1Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
The Journal of Biological Chemistry
|June 12, 2009
Summary
Y-family DNA polymerase Dpo4 exhibits distinct conformational changes during nucleotide selection. Accurate base pairing, not mispairing, triggers specific structural flexibility changes crucial for DNA replication fidelity.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Y-family DNA polymerases are crucial for DNA repair and replication.
- Understanding nucleotide selection mechanisms in these polymerases is key to their function.
- Previous studies faced challenges in identifying structural changes linked to polymerase fidelity.
Purpose of the Study:
- To investigate the conformational dynamics of the Y-family DNA polymerase Dpo4.
- To identify structural changes associated with nucleotide selection and catalytic steps.
- To develop a comprehensive model for Dpo4's nucleotide selection mechanism.
Main Methods:
- Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to monitor protein structure.
- Analysis of Dpo4 conformational changes with and without substrates and ligands.
- Integration of HDX-MS data with previous kinetic studies using fluorescent probes.
Main Results:
- HDX-MS revealed previously unidentified structural changes during Dpo4's catalytic cycle.
- Specific regions, like the alphaB-loop-alphaC and H-helix, showed altered flexibility and accessibility.
- Decreased deuterium exchange correlated with accurate nucleotide insertion and Watson-Crick base pairing.
Conclusions:
- Dpo4's nucleotide selection involves dynamic structural rearrangements.
- Accurate base pairing triggers specific conformational changes essential for fidelity.
- A combined structural and kinetic model explains Dpo4's mechanism of action.
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