How E. coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides.


Deoxy- and dideoxynucleotides differ only in whether they have a ...
Deoxy- and dideoxynucleotides differ only in whether they have a hydroxyl substituent at C-3' of the ribose moiety, and yet the Klenow fragment DNA polymerase prefers the natural (dNTP) substrate by several thousandfold. We have used this preference in order to investigate how Klenow fragment interacts with the sugar portion of an incoming dNTP. We screened mutant derivatives of Klenow fragment so as to identify those amino acid residues that play important roles in distinguishing between dNTPs and ddNTPs. Substitution of Phe762 with Ala or Tyr caused a dramatic decrease in the discrimination against ddNTPs, while mutations in Tyr766 and Glu710 had a smaller effect, suggesting that these two side-chains play secondary roles in the selection of dNTPs over ddNTPs. In order to understand the interactions in the enzyme-DNA-dNTP ternary complex, pre-steady-state kinetic parameters for the incorporation of dNTPs and ddNTPs were determined for wild-type Klenow fragment and for mutant derivatives that showed changes in dNTP/ddNTP discrimination. From elemental effect measurements we infer that selection against dideoxynucleotides takes place in the transition state for the conformational change that precedes phosphoryl transfer. The crucial role of the Phe762 side-chain appears to be to constrain the dNTP molecule so that the 3'-OH can make an interaction with another group within the ternary complex. When Tyr is substituted at position 762, the same interactions can take place to position the dNTP, but specificity against the ddNTP is lost because the phenolic OH can compensate for the missing 3'-OH of the nucleotide. Substitution of the smaller Ala side-chain results in a loss in specificity because the dNTP is no longer appropriately constrained. Measurement of reaction rates as a function of magnesium ion concentration suggests that the interaction made with the dNTP 3'-OH may involve a metal ion and the Glu710 side-chain, the simplest scenario being that both the 3'-OH and the carboxylate of Glu710 are ligands to the same metal ion.



Nucleotide Analogs / Template Lesions, Nucleotide Incorporation


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