Use of Fluorescence Resonance Energy Transfer To Investigate the Conformation of DNA Substrates Bound to the Klenow Fragment ² W. Scott Furey, ‡ Catherine M. Joyce, § Mark A. Osborne, ‡ David Klenerman, ‡ James A. Peliska, ⊥ and Shankar Balasubramanian* ,‡ UniVersity Chemical Laboratory, Cambridge UniVersity, Lensfield Road, Cambridge CB2 1EW, U.K., Department of Molecular Biophysics and Biochemistry, Yale UniVersity, New HaVen, Connecticut 06520-8114, and Department of Biological Chemistry, UniVersity of Michigan Medical School, Ann Arbor, Michigan 48109 ReceiVed August 11, 1997; ReVised Manuscript ReceiVed NoVember 11, 1997 ABSTRACT: Fluorescence resonance energy transfer (FRET) has been used to investigate the conformation of the single stranded region for a series of fluorescent DNA template-primers bound to the Klenow fragment (KF) of Escherichia coli DNA polymerase I. Fluorescent derivatives of template-primer DNA, modified with tetramethylrhodamine (TMR), served as energy transfer acceptors to the donor fluorescein fluorophore used to modify cysteine 751 in the double mutant KF (S751C, C907S). Design of the template-primer allowed the probe’s position within the DNA-protein complex to be varied by stepwise extension of the primer strand upon addition of the appropriate deoxynucleoside triphosphates (dNTP). The TMR acceptor probe occupied seven different positions in the template-primers, five in the single stranded region and two in the double stranded region. The efficiency of energy transfer was determined at each position by calculating the integrated area of the fluorescein emission peak in the presence and absence of acceptor. Results indicate that the FRET efficiency varied in a sinusoidal fashion with a periodicity of approximately 10 base pairs and that the data could be fitted to an equation derived from a simple model formulated on the basis of helical structure. The data support the conclusion that the single stranded template portion of a DNA template-primer adopts a helical conformation when 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. Escherichia coli DNA polymerase I (Pol I) 1 is a multi- functional enzyme responsible for DNA repair and replication in vivo (1). In addition to the 5′-3′ polymerase activity of the enzyme, the separate 3′-5′ and 5′-3′ exonuclease activities are all found on a single 103 kDa polypeptide chain. The large 68 kDa proteolytic fragment of Pol I, termed the Klenow fragment, retains the polymerase and 3′-5′ exonu- clease activities. The Klenow fragment has served as a prototype system for more complex polymerases. Since the original crystal structure was obtained (2), the Klenow fragment has been studied intensely by a variety of structural (e.g. 3-5), kinetic (e.g. refs 6-8), genetic (e.g. refs 9-12), and spectroscopic (e.g. 13 and 14) techniques in order to gain insight into polymerase mechanisms and DNA-protein interactions in such systems. There has recently been a significant improvement in our knowledge of the structures of template-directed polynucleo- tide polymerases and their interactions with nucleic acid substrates. Crystal structures of seven nucleic acid polym- erases are now available: the Klenow fragment of E. coli DNA polymerase I (KF) (2-5), HIV-1 reverse transcriptase (RT) (15-17), Moloney murine leukemia virus reverse transcriptase (18), bacteriophage T7 RNA polymerase (19), rat DNA polymerase  (20, 21), Taq polymerase (22-24), and a thermostable Bacillus DNA polymerase I (25). The most recent crystal structure of Taq polymerase complexed with duplex DNA (22) supports the conclusions of Steitz and co-workers (3) and would appear to have resolved the debate regarding the orientation of DNA binding (26-28). On the basis of the structural homology between Taq po- lymerase and the KF, it would appear reasonable to assume that the KF binds the duplex region in the same manner. Some of these structural studies have involved polymerase- nucleic acid cocrystal structures (3, 5, 15, 17, 21, 22) and have addressed the issue of polymerase-nucleic acid inter- actions. One issue which has not been addressed in detail ² S.B. is a Royal Society Research Fellow. This work was supported by an NSERC 1967 fellowship to W.S.F. and NIH Grant GM-28550 to C.M.J. * To whom correspondence should be addressed at the Cambridge University. Phone: +44-1223-336347. Fax: +44-1223-336913. E- mail: sb10031@cam.ac.uk. ‡ Cambridge University. § Yale University. ⊥ University of Michigan. 1 Abbreviations: Pol I, Escherichia coli DNA polymerase I; KF, Klenow fragment; KF*, fluorescein modified Klenow fragment; TMR, tetramethylrhodamine; dNTP, deoxynucleoside 5′-triphosphate; bp, base pair; kDa, kilodalton; Da, dalton; FRET, fluorescence resonance energy transfer; ESMS, electrospray mass spectrometry; HPLC, high-pressure liquid chromatography; fwhm, full width at half-maximum; TEAB, triethylammonium bicarbonate; TEA, triethylamine; EDTA, ethylene- diaminetetraacetic acid; DTT, dithiothreitol; DMF, dimethylformamide; IPA, 2-propanol; Tris-HCl, tris(hydroxymethyl)aminomethane hydro- chloride; KP i, potassium dihydrogen phosphate/dipotassium hydrogen phosphate; RT, reverse transcriptase; 35*, a TMR labeled 35 mer template oligonucleotide (see Figure 1); 38*, a TMR labeled 38 mer template oligonucleotide (see Figure 1). 2979 Biochemistry 1998, 37, 2979-2990 S0006-2960(97)01975-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/10/1998