Abstract
The mechanism of nucleotide selection by Y-family DNA polymerases has been the subject of intense study, but significant structural contacts and/or conformational changes that relate to polymerase fidelity have been difficult to identify. Here we report on the conformational dynamics of a model Y-family polymerase Dpo4 from Sulfolobus solfataricus. Hydrogen-deuterium exchange in tandem with mass spectrometry was used to monitor changes in Dpo4 structure as a function of time and the presence or absence of specific substrates and ligands. Analysis of the data revealed previously unrecognized structural changes that accompany steps in the catalytic cycle leading up to phosphoryl transfer. For example, the solvent accessibility of the alphaB-loop-alphaC region in the finger domain decreased in the presence of all four dNTP insertion events, but the rate of deuterium exchange, an indicator of conformational flexibility, only decreased during an accurate insertion event. Of particular note is a change in the region surrounding the H-helix of the thumb domain. Upon binding DNA and Mg2+, the H-helix showed a decrease in solvent accessibility and flexibility that was relaxed only upon addition of dCTP, which forms a Watson-Crick base pair with template dG and not during mispairing events. The current study expands upon a previous report from our group that used a fluorescent probe located near the thumb domain to measure the kinetic properties of Dpo4 conformational changes. We now present a model for nucleotide selection by Dpo4 that arises from a synthesis of both structural and kinetic data.
Highlights
The endpoint of each nucleotide selection event cannot be determined solely by the thermodynamics of a given base pair [1, 2]
The Sulfolobus solfataricus DNA polymerase Dpo4 has served as the prototypical Y-family polymerase, because it is especially amenable to structural analysis and it shares many features and properties of other Y-family members
In the present study we sought to identify structural changes that occur during the Dpo4 reaction cycle by using hydrogen-deuterium exchange in tandem with mass spectrometry (HDX-MS)
Summary
The endpoint of each nucleotide selection event cannot be determined solely by the thermodynamics of a given base pair [1, 2]. The molecular features intrinsic to DNA polymerases are generally thought to guide the free-energy landscape of phosphoryl transfer toward the selection of “WatsonCrick” pairs [3, 4], at least among the four canonical bases. The Y-family DNA polymerases (pols) represent one class of polymerases with distinctive structural and functional features [8, 9]. Like most other DNA polymerases, the Y-family pols have three domains: the finger for dNTP selection, the palm for catalysis, and the thumb for double strand DNA contact/orientation, which together form a structure that has been likened to a right hand. In the present study we sought to identify structural changes that occur during the Dpo reaction cycle by using hydrogen-deuterium exchange in tandem with mass spectrometry (HDX-MS). Our results provide new insight into Y-family polymerase catalysis, which may lead to a better understanding of how these enzymes select nucleotide substrates and, how they contribute to mutagenesis
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