A Single Highly Mutable Catalytic Site Amino Acid Is Critical for DNA Polymerase Fidelity

151

118

The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template͞primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wildtype enzyme incorporates dNTP substrates.

Section: Discussionmentioning

confidence: 99%

Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase

Xia

Chen

Sera

et al. 2002

151

118

“…A second possible premutagenic lesion contains an apyrimidinic (AP) site on one strand caused by removal of a thymine at a GT mismatch by a glycosylase (16). Taq polymerase is known to pause significantly opposite an abasic site (29) and primers with an internal abasic site can be extended but the extension products are poorly copied during PCR (30). Also, exposure to high temperatures during PCR may lead to strand breaks at abasic sites.…”

Section: Discussionmentioning

confidence: 99%

The observed human sperm mutation frequency cannot explain the achondroplasia paternal age effect

Tiemann‐Boege

Navidi²,

Grewal³

et al. 2002

152

133

The lifelong spermatogonial stem cell divisions unique to male germ cell production are thought to contribute to a higher mutation frequency in males. The fact that certain de novo human genetic conditions (e.g., achondroplasia) increase in incidence with the age of the father is consistent with this idea. Although it is assumed that the paternal age effect is the result of an increasing frequency of mutant sperm as a man grows older, no direct molecular measurement of the germ-line mutation frequency has been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the nucleotide substitution in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. Surprisingly, the magnitude of the increase in mutation frequency with age appears insufficient to explain why older fathers have a greater chance of having a child with this condition. A number of alternatives may explain this discrepancy, including selection for sperm that carry the mutation or an age-dependent increase in premutagenic lesions that remain unrepaired in sperm and are inefficiently detected by the PCR assay.

“…2) plays an important role both in fidelity and in excluding ribonucleotide residues from the polymerase active site (39,40).…”

Section: Discussionmentioning

confidence: 99%

Discrimination against purine–pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation

Minnick

Liu

Grindley

et al. 2002

We previously identified five derivatives of Klenow fragment DNA polymerase that have lower fidelity because of amino acid substitutions in the polymerase active site. One of these has alanine substituted for the invariant Glu-710, whose side chain interacts with the deoxyribose of the incoming dNTP. Here we show that the E710A enzyme has reduced fidelity for five of the 12 possible mismatches. All but one of these involve misinsertion of pyrimidines, including two transition mismatches A-dCTP and G-dTTP. In contrast, E710A polymerase error rates for the reciprocal C-dATP and T-dGTP transition mismatches were similar to those of the wild-type enzyme. The kinetics of formation of correct base pairs and transition mismatches by the wild-type and E710A polymerases, combined with information on the structure of the DNA polymerase active site and the asymmetry of wobble base pairs, provides a plausible explanation for the differential effects of the E710A mutation on fidelity. The data suggest that the Glu-710 side chain plays a pivotal role in excluding wobble base pairs between template pyrimidines and purine triphosphates by steric clash. Moreover, this same side chain enhances the stability of incoming correct dNTPs, such that loss of this interaction on removal of the side chain leads to lower selectivity against mismatches involving incoming pyrimidines.