Isolation, characterization, and kinetic properties of truncated forms of T4 DNA polymerase that exhibit 3'-5' exonuclease activity.
Limited proteolysis of T4 DNA polymerase generated a 45-kDa and 35-kDa protein complex, which had 3'-5' exonucleolytic activity but lacked polymerase activity. After partial chymotryptic digestion of the cloned and expressed 45-kDa protein derived from T4 DNA polymerase, we isolated a 27-kDa fragment (residues 96-331) that still had 3'-5' exonuclease activity, thus demonstrating that the amino acid residues required for catalysis are included within this fragment. We also show that the apparent Km values for the 3'-5' exonuclease activity exhibited by the 27-kDa fragment are considerably greater than the apparent Km values determined for the intact DNA polymerase on deoxyoligonucleotide substrates having more than 3 bases. In contrast, the kcat values for phosphodiester bond hydrolysis of 3'-terminal nucleotides are not very different when comparing intact T4 DNA polymerase with the 27-kDa fragment derived from it. Thus, participation of residues distal to 331 are not required for catalysis, but only for binding, and, based on the similarity of kcat values, the geometry of the residues responsible for catalysis are preserved even in the absence of the carboxyl-terminal 567 residues.
Lin
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TC
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520.
Karam
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Konigsberg
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D017122:Bacteriophage T4 / Q000201:enzymology*; D002918:Chymotrypsin / Q000494:pharmacology; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014764:Viral Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D002918:Chymotrypsin