PEPTIDE RESCUE OF AN N-TERMINAL TRUNCATION OF THE STOFFEL FRAGMENT OFTAQ DNA-POLYMERASE

Deletion of the first 289 amino acids of the DNA polymerase from Therm us aquaticus (Tag polymerase) removes the 5' to 3' exonuclease domain to yield the thermostable Stoffel polymerase fragment (Lawyer et al., 1989). Preliminary N-terminal truncation studies of the Stoffel fragme nt suggested that removal of an additional 12 amino acids (the Stof De lta 12 mutant) had no significant effect on activity or stability, but that the further truncation of the protein (the Stof Delta 47, in whi ch 47 amino acids were deleted), resulted in a significant loss of bot h activity and thermostability. A 33-amino acid synthetic peptide, bas ed on this critical region (i.e., residues 303-335 inclusive), was abl e to restore 85% of the Stof Delta 12 activity when added back to the truncated Stof Delta 47 protein as well as return the temperature opti mum to that of the Stof Delta 12 and Stoffel proteins. Examination of the crystal structure of Tag polymerase (Kim et al., 1995) shows that residues 302-336 of the enzyme form a three-stranded beta-sheet struct ure that interacts with the remainder of the protein. CD analysis of t he 33-amino acid peptide indicates that the free peptide also adopts a n ordered structure in solution with more than 50% beta-sheet content. These data suggest that this 33-amino acid peptide constitutes a stab le beta-sheet structure capable of rescuing the truncated polymerase i n a fashion analogous to the well-documented complementation of Ribonu clease S protein by the 15-residue, alpha-helical, S peptide.