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Dissecting changes in Dpo4 DNA polymerase structure during catalysis by hydrogen‐deuterium exchange mass spectrometry
Author(s) -
Eoff Robert Lawton,
SanchezPonce Raymundo,
Guengerich Frederick Peter
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.990.1
Subject(s) - sulfolobus solfataricus , hydrogen–deuterium exchange , chemistry , polymerase , dna polymerase , catalysis , dna , biochemistry , mass spectrometry , archaea , chromatography , gene
Sulfolobus solfataricus DNA polymerase IV (Dpo4) has been studied in vitro as a model for Y‐family polymerase catalysis. Some initial kinetic studies with Dpo4 suggested that a non‐covalent step precedes the phosphoryl transfer step in the reaction cycle, which conforms to the “induced‐fit” mechanism for polymerase catalysis. Such a step is often attributed to conformational changes in the enzyme structure, but x‐ray crystal structures of Dpo4 in various catalytic states have failed to reveal the large domain rearrangements that have been observed for polymerases such as bacteriophage T7 DNA polymerase. Our group has used hydrogen‐deuterium exchange in tandem with mass spectrometry in an attempt to resolve structural changes that occur during Dpo4 catalysis. Distinct structural changes were observed upon binding to DNA and upon inclusion of a divalent metal ion. Analysis of these changes may provide insight into non‐covalent steps that define reaction kinetics and contribute to polymerase fidelity.