The fluorescence of 2-aminopurine deoxynucleotide positioned in a 3-terminal mismatch was used to evaluate the pre-steady state kinetics of the 3 3 5 exonuclease activity of bacteriophage T4 DNA polymerase on defined DNA substrates. DNA substrates with one, two, or three preformed terminal mispairs simulated increasing degrees of strand separation at a primer terminus. The effects of base pair stability and local DNA sequence on excision rates were investigated by using DNA substrates that were

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... either relatively G ؉ C-or A ؉ T-rich. The importance of strand separation as a prerequisite to the hydrolysis of a terminal nucleotide was demonstrated by using a unique mutant DNA polymerase that could degrade single-stranded but not double-stranded DNA, unless two or more 3-terminal nucleotides were unpaired. Our results led us to conclude that the reduced exonuclease activity of this mutant DNA polymerase on duplex DNA substrates is due to a defect in melting the primer terminus in preparation for the excision reaction. The mutated amino acid (serine substitution for glycine at codon 255) resides in a critical loop structure determined from a crystallographic study of an amino-terminal fragment of T4 DNA polymerase. These results suggest an active role for amino acid residues in the exonuclease domain of the T4 DNA polymerase in the strand separation step. Exonucleolytic proofreading by DNA polymerases is an important component of high fidelity DNA replication. Nucleotide insertion errors occur at a frequency of 10 Ϫ3 to 10 Ϫ5 , but the DNA polymerase-associated 3Ј 3 5Ј-exonuclease activity further reduces replication errors by 100-fold or more (reviewed in Ref. 1). Different steps of the proofreading reaction have been revealed by mutational analysis of the bacteriophage T4 DNA polymerase. Mutant T4 DNA polymerases deficient in 3Ј 3 5Ј-exonuclease activity exhibit a strong mutator phenotype that can be used to select for mutant DNA polymerases (2-4). The loss of the 3Ј 3 5Ј-exonuclease activity by mutation can be achieved by preventing distinct steps in the proofreading process. The hydrolysis reaction may be prevented if residues required for catalysis are substituted with other amino acids. For example, the substitution of Ala residues for Asp residues that bind essential Mg 2ϩ ions in the exonuclease active center re-duces the exonuclease activity to barely detectable levels (5-7). Another reaction step in proofreading that may be affected by mutation is the translocation of the primer end of the DNA between the spatially distinct polymerase and exonuclease active centers. Identification of several "active-site switching mutants" has been described recently (8, 9) . DNA polymerase proofreading is a dynamic process; if "switching" to the exonuclease active site is reduced by mutation, there is less opportunity for proofreading and a mutator phenotype is produced (9). Another proofreading reaction step that may be probed by mutational analysis of T4 DNA polymerase is strand separation. The 3Ј 3 5Ј-exonuclease activity of T4 DNA polymerase (10) and other proofreading DNA polymerases is a singlestranded DNA exonuclease activity. Structural studies of the Klenow fragment (KF) 1 of Escherichia coli DNA polymerase I show that single-stranded DNA is bound in the exonuclease active center (11), and time-resolved fluorescence experiments confirm that single-stranded DNA is bound under physiological conditions and in an extended conformation (12). Cross-linking experiments demonstrate that at least four 3Ј-terminal nucleotides must be separated from the template strand in order for KF to excise the terminal nucleotide and that a two nucleotide strand separation is required for T4 DNA polymerase (13). The mechanism, however, for converting the natural substrate for proofreading, a duplex DNA with a terminal mismatch, into a DNA with a strand separation of two or more nucleotides has not been elucidated. We report here the biochemical characterization of a mutant T4 DNA polymerase with an apparent defect in strand separation. This mutant, the G255S-DNA polymerase (Gly to Ser substitution at codon 255), was isolated by two different genetic selection strategies for mutant DNA polymerases with reduced exonucleolytic proofreading. In one method, the mutant was identified on the basis of a strong mutator phenotype (2). In the second method, mutations that encode G255A (Ala substitution for Gly-255) and G255S amino acid substitutions were identified repeatedly as suppressors of the excessive proofreading produced by other DNA polymerase mutations (9). Residue Gly-255 and the surrounding region has been identified as a "hot spot" by our genetic selection methods for mutations that decrease proofreading (9). Although it has only been assumed that Gly-255 resides in the exonuclease domain based on protein sequence comparisons to KF (2-5), recent structural studies of an amino-terminal fragment of the T4 DNA polymerase verify that residue Gly-255 is located in the exonuclease domain (14) . Because of the numerous independent genetic isolations of