USE OF FLUORESCENCE RESONANCE ENERGY-TRANSFER TO INVESTIGATE THE CONFORMATION OF DNA SUBSTRATES BOUND TO THE KLENOW FRAGMENT

Fluorescence resonance energy transfer (FRET) has been used to investi gate the conformation of the single stranded region for a series of fl uorescent DNA template-primers bound to the Klenow fragment (KF) of Es cherichia coli DNA polymerase I. Fluorescent derivatives of template-p rimer DNA, modified with tetramethylrhodamine (TMR), served as energy transfer accepters to the donor fluorescein fluorophore used to modify cysteine 751 in the double mutant KF (S751C, C907S). Design of the te mplate-primer allowed the probe's position within the DNA-protein comp lex to be varied by stepwise extension of the primer strand upon addit ion of the appropriate deoxynucleoside triphosphates (dNTP). The TMR a cceptor probe occupied seven different positions in the template-prime rs, five in the single stranded region and two in the double stranded region. The efficiency of energy transfer was determined at each posit ion by calculating the integrated area of the fluorescein emission pea k in the presence and absence of acceptor. Results indicate that the F RET efficiency varied in a sinusoidal fashion with a periodicity of ap proximately 10 base pairs and that the data could be fitted to an equa tion derived from a simple model formulated on the basis of helical st ructure. The data support the conclusion that the single stranded temp late portion of a DNA template-primer adopts a helical conformation wh en bound to the KF. The results of this study further support FRET as a useful method for the determination of structure and conformation in protein-DNA complexes.