MELTING OF A DNA HELIX TERMINUS WITHIN THE ACTIVE-SITE OF A DNA-POLYMERASE

Accurate synthesis of DNA by polymerases is due in part to the selecti ve removal of misincorporated nucleotides by a 3'-5' exonuclease activ ity (proofreading). Proofreading by an exonuclease domain containing a single-stranded DNA binding site may involve local melting of a duple x DNA substrate. Here we use time-resolved fluorescence spectroscopy t o analyze the local melting of a DNA duplex terminus induced by the Kl enow fragment of DNA polymerase I. Four oligodeoxynucleotide primer/te mplates were prepared, each containing the fluorescent adenine analog 2-aminopurine (A) at the primer 3' terminus, and one of the common DN A bases opposite the A residue. Fluorescence decays of the duplex DNA s and the single primer oligonucleotide were jointly analyzed using gl obal analysis procedures. Four lifetime components were resolved in th e duplex DNAs, representing distinct conformational states of the term inal A residue: paired A* bases, partially stacked A* bases, and exte nded A bases. The variation of the apparent fraction of paired A* bas es with temperature was in accord with optical melting data, and the e xtent of base pairing observed in each duplex was consistent with the base-pairing preferences of A established in other studies. These res ults establish that the fluorescence decay characteristics of A can b e 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 D NA melting transitions, this method was used to examine the extent to which Klenow fragment could induce fraying at each duplex terminus. Ad dition of D424A mutant Klenow fragment to the primer/template DNAs dec reased the fraction of paired terminal bases and increased the fractio n of extended terminal bases. These results demonstrate that Klenow fr agment can melt a DNA duplex terminus, resulting in a population of DN A molecules in which the primer terminus is bound in an extended singl e-stranded conformation. The results of this study also establish a ne w method for examining base-pairing interactions within DNA molecules bound to proteins.