HOW ESCHERICHIA-COLI DNA-POLYMERASE-I (KLENOW FRAGMENT) DISTINGUISHESBETWEEN DEOXYNUCLEOTIDES AND DIDEOXYNUCLEOTIDES

Deoxy- and dideoxynucleotides differ only in whether they have a hydro xyl substituent at C-3' of the ribose moiety, and yet the Klenow fragm ent DNA polymerase prefers the natural (dNTP) substrate by several tho usandfold. We have used this preference in order to investigate how Kl enow fragment interacts with the sugar portion of an incoming dNTP. We screened mutant derivatives of Klenow fragment so as to identify thos e amino acid residues that play important roles in distinguishing betw een dNTPs and ddNTPs. Substitution of Phe762 with Ala or Tyr caused a dramatic decrease in the discrimination against ddNTPs, while mutation s in Tyr766 and Glu710 had a smaller effect, suggesting that these two side-chains Flay secondary roles in the selection of dNTPs over ddNTP s. In order to understand the interactions in the enzyme-DNA-dNTP tern ary 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 discriminati on. From elemental effect measurements we infer that selection against dideoxynucleotides takes place in the transition state for the confor mational 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 int eractions can take place to position the dNTP, but specificity against the ddNTP is lost because the phenolic OH can compensate for the miss ing 3'-OH of the nucleotide. Substitution of the smaller Ala side-chai n results in a loss in specificity because the dNTP is no longer appro priately constrained. Measurement of reaction rates as a function of m agnesium ion concentration suggests that the interaction made with the dNTP 3'-OH may involve a metal ion and the Glu710 side-chain, the sim plest scenario being that both the 3'-OH and the carboxylate of Glu710 are ligands to the same metal ion. (C) 1998 Academic Press Limited.