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United States Department of Agriculture

Agricultural Research Service

Title: Use of Viscogens, Dntpas, and Rhodium(iii) As Probes in Stopped-Flow Experiments to Obtain New Evidence for the Mechanism of Catalysis by DNA Polymerase Beta

item Bakhtina, Marina - OH STATE UNIV, COLUMBUS
item Lee, Soojin - OH STATE UNIV, COLUMBUS
item Dunlap, Christopher
item Lamarche, Brandon - OH STATE UNIV, COLUMBUS

Submitted to: Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 5, 2005
Publication Date: March 10, 2005
Citation: Bakhtina, M., Lee, S., Wang, Y., Dunlap, C.A., Lamarche, B., Tsai, M. 2005. Use of viscogens, dNTP-alpha-S, and Rhodium(III) as probes in stopped-flow experiments to obtain new evidence for the mechanism of catalysis by DNA polymerase beta. Biochemistry. 44(13):5177-5187.

Interpretive Summary: DNA polymerases are responsible for the replication and repair of genomic information. The damage and mutation of DNA is the leading cause of cancer and related diseases. Understanding how polymerases function provides insights into the fundamental causes of these diseases. The current study addresses how DNA polymerases are able to faithfully repair or replicate DNA without making mistakes. The work generates new data to support a mechanism of fidelity for DNA polymerases. This work will benefit medical scientists and biochemists trying to understand the molecular basis for enzyme catalysis.

Technical Abstract: The kinetic mechanism and the structural bases of the fidelity of DNA polymerases are still highly controversial. Here we report the use of three probes in the stopped-flow studies of polymerase-beta to obtain new, direct evidence for our previous interpretations: (a) Increasing the viscosity of the reaction buffer by sucrose or glycerol is expected to slow down the conformational change differentially, and it was shown to slow down the first (fast) fluorescence transition selectively. (b) Use of deoxynucleotriphosphate (dNTP) sulfur analogues in place of native dNTP is expected to slow down the chemical step preferentially, and it was shown to slow down the second (slow) fluorescence transition selectively. (c) The substitution-inert Rhodium (III) dNTP was used to show for the first time that the slow fluorescence change occurs after mixing of Pol/DNA/Rhodium (III) dNTP with Magnesium (II). These results, along with crystal structures, suggest that the subdomain-closing conformational change occurs before binding of the catalytic Magnesium (II) while the rate-limiting step occurs after binding of the catalytic Magnesium (II). These results provide new evidence to the mechanism we suggested previously, but do not support the results of three recent papers of computational studies. The results were further supported by a “sequential mixing” stopped-flow experiment that used no analogues, and thus ruled out the possibility that the discrepancy between experimental and computational results is due to the use of analogues. The methodologies can be used to examine other DNA polymerases to answer whether the properties of Pol beta are exceptional or general.

Last Modified: 4/21/2015