A thumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity.

Abstract:

In Klenow fragment DNA polymerase, a flexible 50-amino acid subdomain at the tip of the thumb which includes two alpha helices has been suggested to interact with the duplex template-primer (Beese, L.S., Derbyshire, V. and Steitz, T.A. (1993) Science 260, 352-355). The present study investigates the properties of Klenow polymerase containing a 24-amino acid deletion (residues 590-613) that removes a portion of the tip of the thumb. The mutant polymerase has relatively normal dNTP binding and catalytic rate. However, its DNA binding affinity is reduced by more than 100-fold relative to the intact polymerase and its ability to conduct processive synthesis is also reduced. Although the mutant polymerase has relatively normal base substitution fidelity, it has strongly reduced frameshift fidelity, being especially error-prone for single nucleotide addition errors in homopolymeric runs. The addition error rate increases as the length of the reiterated sequence increases, indicative of errors initiated by template-primer strand slippage. These observations suggest a role for the tip of the thumb of Klenow polymerase in determining DNA binding, processivity and frameshift fidelity, perhaps by tracking the minor groove of the duplex DNA. The results are discussed in light of remarkably similar observations with T7 DNA polymerase in the presence or absence of thioredoxin, an accessory subunit that affects these same properties.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Source / Purification, Fidelity, Kinetic Parameters

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow fragment Minnick DT1996 Molecular Weight 6.8E+04 Dalton
Klenow fragment Minnick DT1996 Cloned or native Native organism
Klenow fragment Minnick DT1996 Tagged No
Klenow fragment Minnick DT1996 Frameshift Error Rate 0.0102 errors/bp Technique: Reversion
Klenow fragment Minnick DT1996 Overall Error Rate 4E-05 errors/bp
Klenow fragment Minnick DT1996 Nucleotide Substitution Rate 0.00036 errors/bp Technique: Reversion
Klenow fragment Minnick DT1996 Nucleotide Substitution Rate 0.0028 errors/bp Technique: Forward mutational
Klenow fragment Minnick DT1996 Full length or truncated Full length
Klenow fragment Minnick DT1996 KM 3.7uM Reaction: Nucleotide incorporation; Substrate: dTTP
Klenow fragment Minnick DT1996 Kd 42nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Klenow fragment Minnick DT1996 kcat 0.3 /second Reaction: Nucleotide incorporation; Substrate: dTTP

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