Isolation, characterization, and kinetic properties of truncated forms of T4 DNA polymerase that exhibit 3'-5' exonuclease activity.

Abstract:

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.

Polymerases:

Topics:

Kinetic Parameters, Structure and Structure/Function, Nucleotide Incorporation, Exonuclease Activity, Methods

One line summary:

Using proteolytic fragments of T4 pol to determine structure/function

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow fragment Lin TC1994 KM 3uM Reaction: 3-5' Exonuclease; Substrate: DNA template
Klenow fragment Lin TC1994 KM 160uM Reaction: 3-5' Exonuclease; Substrate: DNA template
Klenow fragment Lin TC1994 kcat 5.8 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
Klenow fragment Lin TC1994 kcat 7.8 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 Lin TC1994 3-5' Exonuclease (proofreading) Yes
T4 Lin TC1994 KM 1000uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 Lin TC1994 KM 7uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 Lin TC1994 kcat 170 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 Lin TC1994 kcat 420 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 N698 Lin TC1994 KM 3300uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 N698 Lin TC1994 KM 110uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 N698 Lin TC1994 kcat 170 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 N698 Lin TC1994 kcat 270 /second Reaction: 3-5' Exonuclease
T4 N388 Lin TC1994 KM 3800uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 N388 Lin TC1994 KM 1000uM Substrate: DNA template
T4 N388 Lin TC1994 kcat 170 /second Reaction: 3-5' Exonuclease; Substrate: DNA template

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