Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• DNA proofreading by DNA polymerase I is essential for maintaining genomic integrity.
• This process requires translocation of a damaged primer terminus from the polymerase active site to the exonuclease active site.
• The precise mechanism of this translocation remains incompletely understood.

Purpose of the Study:

• To elucidate the mechanism of DNA primer terminus transfer during DNA polymerase proofreading.
• To quantify the equilibrium partitioning of DNA substrates between polymerase and exonuclease active sites.
• To identify key factors influencing the proofreading efficiency of DNA polymerase I.

Main Methods:

• Utilized time-resolved fluorescence spectroscopy with dansyl-labeled DNA primer/templates.
• Studied the interaction between DNA substrates and the Klenow fragment of Escherichia coli DNA polymerase I.
• Analyzed fluorescence lifetime and angular diffusion changes to determine DNA substrate location.

Main Results:

• Distinct fluorescence properties of dansyl probes indicated DNA substrate location at either the polymerase or exonuclease site.
• Observed a characteristic 'dip and rise' anisotropy decay when both binding modes were populated.
• Nonlinear least-squares analysis quantified the equilibrium partitioning constant (Kpe) between polymerase and exonuclease sites.

Conclusions:

• The study provides a quantitative method to measure DNA substrate partitioning during proofreading.
• Changes in Kpe reflect alterations in the equilibrium between polymerase and exonuclease binding modes.
• This approach allows identification of determinants affecting DNA polymerase proofreading efficiency.