USING 2-AMINOPURINE FLUORESCENCE AND MUTATIONAL ANALYSIS TO DEMONSTRATE AN ACTIVE-ROLE OF BACTERIOPHAGE-T4 DNA-POLYMERASE IN STRAND SEPARATION REQUIRED FOR 3'-]5'-EXONUCLEASE ACTIVITY

The fluorescence of S-aminopurine deoxynucleotide positioned in a 3'-t erminal mismatch was used to evaluate the pre-steady state kinetics of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase on defined DNA substrates, DNA substrates with one, two, or three pre formed terminal mispairs simulated increasing degrees of strand separa tion at a primer terminus. The effects of base pair stability and loca l DNA sequence on excision rates were investigated by using DNA substr ates that were 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 t hat 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 p olymerase on duplex DNA substrates is due to a defect in melting the p rimer terminus in preparation for the excision reaction, The mutated a mino 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 a n active role for amino acid residues in the exonuclease domain of the T4 DNA polymerase in the strand separation step.