We have identified an amino-proximal sequence motif, Phe-Asp-lle-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3' --5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolinsensitive DNA polymerases possessing 3' -*5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase Il had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3' -* 5' exonuclease active site ofE. coiU DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3' -5' proofreading exonuclease activity. None ofthese residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila a DNA polymerases.Class B DNA polymerasest are related through a series of conserved amino acid sequences that occur in the order IV, II, VI, III, I, V, with region I being the most highly conserved (2). Regions II, III, I, and V are implicated in DNA polymerase functions (3)(4)(5)(6)(7)(8). The location of the 3' -* 5' exonuclease activity domain has been sought through amino acid sequence similarity with the apparently unrelated E. coli DNA polymerase I. Crystallographic and mutational evidence has determined that the 3' -*5' exonuclease active site of E. coli DNA polymerase I is composed of a group of carboxylate residues (D355, E357, D424, and D501) clustering around two metal ions that coordinate the 3'-terminal phosphate and a second group of residues (L361, F473, and Y497) located around the terminal base and ribose positions (9, 10). It has been proposed that residues D355 and E357 are conserved in the left part of region IV ofaphidicolin-sensitive DNA polymerases (11-13). Similarly, residues D424, Y497, and D501 were suggested to be conserved in the right part of, and distal to, region IV (4,12,13). Mutation to alanine of three of the relevant carboxylate residues in bacteriophage 029 DNA polymerase leads to exonuclease-deficient DNA polymerase activity (13). Though related to other aphidicolinsensitive DNA polymerases, 429 DNA polymerase does not have a well-conserved region IV (2) and may not be a good test case for alignments involving this region. With coliphage T4 DNA polymerase, which does have a well-conserved region IV (2, 11, 12), mutation to alanine ofresidues D189 and E191, predicted to correspond to the E. coli polymerase residues D355 and E357, does not lead to exonuclease deficiency, calling into question the concept of a generally conserved 3'-* 5' exonuclease active sit...

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).

A mutant derivative of Klenow fragment DNA polymerase containing serine substituted for tyrosine at residue 766 has been shown by kinetic analysis to have an increased misinsertion rate relative to wild-type Klenow fragment, but a decreased rate of extension from the resulting mispairs (Carroll, S. S., Cowart, M., and Benkovic, S. J. (1991) Biochemistry 30, 804 -813). In the present study we use an M13mp2-based fidelity assay to study the error specificity of this mutator polymerase. Despite its compromised ability to extend mispairs, the Y766S polymerase and a Y766A mutant both have elevated base substitution error rates. The magnitude of the mutator effect is mispair-specific, from no effect for some mispairs to rates elevated by 60-fold for misincorporation of TMP opposite template G. The results with the Y766S mutant are remarkably consistent with the earlier kinetic analysis of misinsertion, demonstrating that either approach can be used to identify and characterize mutator polymerases. Both the Y766S and Y766A mutant polymerases are also frameshift mutators, having elevated rates for two-base deletions and a 276-base deletion between a direct repeat sequence. However, neither mutant polymerase has an increased error rate for single-base frameshifts in repetitive sequences. This error specificity suggests that the deletions generated by the mutator polymerases are initiated by misinsertion rather than by strand slippage. When considered with recent structure-function studies of other polymerases, the data indicate that the nucleotide misinsertion and strand-slippage mechanisms for polymerization infidelity are differentially affected by changes in distinct structural elements of DNA polymerases that share similar subdomain structures.Among the most important properties of a DNA polymerization reaction is its fidelity. Numerous studies (reviewed in Refs. 1-3) have shown that two events initiate most polymerization errors. One is the misinsertion of an incorrect nucleotide. This usually yields a base substitution mutation, but can also yield a frameshift when the misinserted nucleotide is complementary to an adjacent template base and the primer relocates to produce misaligned strands. Several steps in the reaction cycle can affect the rate of errors initiated by misinsertion, including dNTP binding, a subsequent conformational change preceding chemistry and the rate of phosphodiester bond formation. Also critical is the balance between extension of a misinserted base and its exonucleolytic removal or rearrangement. The second error-initiating event is template-primer slippage (Ref. 4, reviewed in Ref. 2), usually resulting in deletion or addition of one or more nucleotides, particularly in repetitive sequences.One approach for understanding these two error-initiating events is to study the properties of DNA polymerases whose x-ray crystal structures have revealed interactions with the template-primer and incoming dNTPs that might influence fidelity. Studies of four polymerases (reviewed in Ref. 5) indicate ...

The enzymatic decarboxylation of orotidine 5'-monophosphate may proceed by an addition-elimination mechanism involving a covalently bound intermediate or by elimination of CO2 to generate a nitrogen ylide. In an attempt to distinguish between these two alternatives, 1-(phosphoribosyl)barbituric acid was synthesized with 13C at the 5-position. Interaction of this potential transition-state analogue inhibitor with yeast orotidine-5'-monophosphate decarboxylase resulted in a small (0.6 ppm) downfield displacement of the C-5 resonance, indicating no rehybridization of the kind that might have been expected to accompany 5,6-addition of an enzyme nucleophile. When the substrate orotidine 5'-monophosphate was synthesized with deuterium at C-5, no significant change in kcat (H/D = 0.99 +/- 0.06) or kcat/KM (H/D = 1.00 +/- 0.06) was found to result, suggesting that C-5 does not undergo significant changes in geometry before or during the step that determines the rate of the catalytic process. These results are consistent with a nitrogen ylide mechanism and offer no support for the intervention of covalently bound intermediates in the catalytic process.