Influence of substitutions at the conserved aromatic amino acid residues around the active site of ExoIII

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism"</p><p>Nucleic Acids Research 2006;34(5):1552-1563.</p><p>Published online 15 Mar 2006</p><p>PMCID:PMC1408312.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Detection of products of cleavage by ExoIII and its mutant proteins. Proteins used in each reaction are shown above each lane. The oligonucleotide containing an AP site (AP-ssDNA) was d-GCGATGACTAACGTACTAGGCTTCCGAGCC. AP-dsDNA was constructed by annealing AP-ssDNA and d-CGGAAGCCTAGTATCGTTAGTCATCGCCATG. The substrate DNA (4.4 pmol), in which the oligonucleotide containing an AP site was 5′ P-labeled, was incubated with the wild-type ExoIII or its mutant (0.04 pmol) in buffer containing 75 mM NaCl, 5 mM MgCl, 10 mM DTT and 65 mM Tris–HCl (pH 8.0) at 23°C. The DNA products were analyzed using a 20% denaturing (7 M urea) polyacrylamide gel. The product 1 was produced by AP endonucleolytic cleavage at the 5′ side of the AP site. The product 2 was produced by exonucleolytic cleavage from 3′ end of the product 1. The markers were 5′ P-labeled d-GCGATGACTAACG. () Abilities of ExoIII and its mutant proteins to bind the dsDNA containing an AP site. The proteins used in each reaction are shown above each lane. AP-dsDNA (0.45 pmol), in which the oligonucleotide containing an AP site was 5′ P-labeled, was incubated with the wild-type ExoIII or its mutant (4.5 pmol) in buffer containing 77 mM NaCl, 10 mM EDTA and 66 mM Tris–HCl (pH 8.3) at 4°C. The protein–DNA complex was analyzed by 15% native polyacrylamide gel electrophoresis