Peptide I, a 50-amino acid synthetic peptide based on residues 728 to 777 of DNA polymerase I, binds dNTP substrates and duplex DNA (G. Mullen, P. Shenbagamurthi, and A.S. Mildvan, J. Biol. Chem. 264, 19637-19647, 1988). The structural properties of peptide I at pH 3.9 have been studied by CD spectroscopy and by 2D proton NMR at 600 MHz. The CD spectra are fit by assuming that peptide I contains 17% helix, 17% beta-structure, and 66% coil. The substrate dATP binds tightly to peptide I under these conditions (KD = 0.5 microM) as determined by fluorescence quenching but induces no change in peptide conformation, as detected by CD spectroscopy. Proton resonances of peptide I have been assigned by double quantum filtered correlated spectroscopy, total correlated spectroscopy, and nuclear Overhauser effect spectroscopy. As found with other peptides, peptide I is best characterized by both extended and partially folded secondary structures which equilibrate rapidly on the NMR time scale. A region from residues 3 through 10 displays nuclear Overhauser effects (NOEs) consistent with the rapid equilibration of a nascent helix with a random extended structure. Alternatively this segment of residues is consistent with a series of three opened-out turns. A nonclassical turn is found between residues 14 and 17 and from residues 44 to 47, the latter closing irregular antiparallel strands from residues 42 to 48. The remainder of the peptide is a coil. A residue-by-residue comparison of the best-fit solution structure of the peptide with that of the corresponding sequence in the X-ray structure of the complete enzyme reveals that 36% of the amino acids are found to be in a conformation similar to that in the enzyme. Such partial and transient folding of the peptide indicates that the major role of the remainder of the protein is to provide structural support for the active site region of the enzyme. As detected by interresidue NOEs and NOEs to water protons, the homologous sequence Leu-37-Ile-38-Tyr-39-Gly-40, together with Phe-15 of the peptide, provides an exposed hydrophobic cluster of residues which may constitute the substrate binding site. An exposed cluster of cationic residues consisting of Arg-27, Arg-28, Lys-31, and possibly Arg-48 may provide the binding site for duplex DNA.