Accurate synthesis of DNA by polymerase is due in part to the selective removal of misincorporated nucleotides by a 3'-5' exonuclease activity (proofreading). Proofreading by an exonuclease domain containing a single-stranded DNA binding site may involve local melting of a duplex DNA substrate. Here we use time-resolved fluorescence spectroscopy to analyze the local melting of a DNA duplex terminus induced by the Klenow fragment of DNA polymerase I. Four oligodeoxynucleotide primer/templates were prepared, each containing the fluorescent adenine analog 2-aminopurine (A*) at the primer 3' terminus, and one of the common DNA bases opposite the A* residue. Fluorescence decays of the duplex DNAs and the single primer oligonucleotide were jointly analyzed using global analysis procedures. Four lifetime components were resolved in the duplex DNAs, representing distinct conformational states of the terminal A* residue: paired A* bases, partially stacked A* bases, and extended A* bases. The variation of the apparent fraction of paired A* bases with temperature was in accord with optical melting data, and the extent of base pairing observed in each duplex was consistent with the base-pairing preferences of A* established in other studies. These results establish that the fluorescence decay characteristics of A* can be used to examine base-pairing interactions at a DNA duplex terminus. Since the fluorescence of A* can be observed without interference from protein amino acid residues, unlike existing methods for monitoring DNA melting transitions, this method was used to examine the extent to which Klenow fragment could induce fraying at each duplex terminus.(ABSTRACT TRUNCATED AT 250 WORDS)