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 ␣ 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 templateprimer 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.The Klenow fragment, 1 a 68-kDa carboxyl-terminal fragment of Escherichia coli DNA polymerase I, contains both DNA polymerase and 3Ј 3 5Ј exonuclease activity. The polymerase active site is on a 45-kDa COOH-terminal domain of Klenow fragment, and the 3Ј 3 5Ј proofreading exonuclease active site is on the smaller NH 2 -terminal domain. Structural information obtained from crystal structures of Klenow fragment demonstrates that the polymerase domain resembles a partially open right hand with three subdomains that form a cleft (for review, see Joyce and Steitz (1994)). The palm is located at the base of the cleft and contains the catalytically important carboxylates. The fingers form one wall of the cleft and the thumb another.

Base Miscoding and Strand Misalignment Errors by Mutator Klenow Polymerases with Amino Acid Substitutions at Tyrosine 766 in the O Helix of the Fingers Subdomain

“…The mutator effects observed here are distinctly different from earlier observations with mutants of HIV-1 reverse transcriptase (8,9) or Klenow fragment (10). Those enzymes are mutators for frameshifts in homopolymeric runs that may be initiated by template-primer slippage, and they contain changes in the thumb subdomain thought to interact with the duplex template-primer.…”

“…Alanine scanning mutagenesis of helix H has identified two mutant reverse transcriptases that have strongly reduced DNA binding affinity, reduced processivity, and reduced fidelity for errors in homopolymeric runs that were consistent with the strand slippage model (8,9). More recently, we have found that a mutant derivative of Klenow fragment polymerase lacking 24 amino acids at the tip of the thumb has reduced DNA binding affinity and also has reduced fidelity for one-base addition and deletion errors in homopolymeric runs (10). These frameshift mutator mutants of HIV-1 reverse transcriptase and Klenow polymerase have relatively normal rates for base substitution errors initiated by nucleotide misinsertion.…”

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

“…However, the loss of DNA binding activity occurred without significant loss of exonuclease activity. Biochemical studies suggest that 5-8 base pairs of duplex DNA are covered by E. coli Klenow fragment when the primer terminus is in the pol active site (33). In the co-crystal structure of an editing complex, the 3Ј terminus is bound in the exo active site, whereas the duplex portion of the template-primer occupies the cleft between exo and pol domains similar to binding in the pol mode (18,40).…”

“…Although we show that our polymerase-defective mutants PKL, GWF, and W576A bind the duplex oligonucleotide substrate poorly, binding to the mismatched DNA substrate is clearly sufficient for these holoenzymes to hydrolyze efficiently a 3Ј-terminal mispaired nucleotide. The earlier study showed that the tip of the thumb subdomain in Klenow is important for template-primer DNA binding and enzyme processivity (33); side chain changes in the thumb also affect the fractional occupancies of template-primer into the two active sites (44). Likewise, amino acid substitutions in the spacer region of the catalytic subunit of pol ␥ may increase the partitioning of the 3Ј terminus to the 3Ј-5Ј exonuclease active site.…”

“…Both biochemical and structural studies of family A DNA polymerases support the possibility that amino acid residues required for enzyme function lie outside the catalytic active site regions (19,(33)(34)(35)(36). Because structural information on pol ␥-␣ is very limited, a secondary structure prediction algorithm (PredictProtein) was used to identify the positions of potential ␣-helices and ␤-strands in the spacer region, and this information was taken into consideration in the generation of triple and nested single alanine mutations that were introduced as substitutions for highly conserved residues in the ␥1, ␥3, and ␥4 elements (Fig.…”