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• W2006737672 endingPage "e1389" @default.
• W2006737672 startingPage "e1389" @default.
• W2006737672 abstract "DNA and RNA polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. We previously showed, for the reverse transcriptase (RT) encoded by the yeast retrotransposon Ty1, that one of the three conserved active site aspartates (D211) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. Transposition is partially restored by second site suppressor mutations in the RNAse H domain. The novel properties of this amino acid substitution led us to express the WT and D211N mutant enzymes, and study their pre-steady state kinetic parameters. We found that the kpol was reduced by a factor of 223 in the mutant, although the Kd for nucleotide binding was unaltered. Further, the mutant enzyme had a marked preference for Mn2+ over Mg2+. To better understand the functions of this residue within the Ty1 RT active site, we have now examined the in vitro properties of WT and D211N mutant Ty1 RTs in carrying out pyrophosphorolysis, the reverse reaction to polymerization, where pyrophosphate is the substrate and dNTPs are the product. We find that pyrophosphorolysis is efficient only when the base-paired primer template region is >14 bases, and that activity increases when the primer end is blunt-ended or recessed by only a few bases. Using pre-steady state kinetic analysis, we find that the rate of pyrophosphorolysis (kpyro) in the D211N mutant is nearly 320 fold lower than the WT enzyme, and that the mutant enzyme has an ∼170 fold lower apparent Kd for pyrophosphate. These findings indicate that subtle substrate differences can strongly affect the enzyme's ability to properly position the primer-end to carry out pyrophosphorolysis. Further the kinetic data suggests that the D211 residue has a role in pyrophosphate binding and release, which could affect polymerase translocation, and help explain the D211N mutant's transposition defect." @default.
• W2006737672 created "2016-06-24" @default.
• W2006737672 creator A5034388856 @default.
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• W2006737672 date "2008-01-02" @default.
• W2006737672 modified "2023-09-26" @default.
• W2006737672 title "Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase" @default.
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• W2006737672 doi "https://doi.org/10.1371/journal.pone.0001389" @default.
• W2006737672 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2148107" @default.
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• W2006737672 hasPublicationYear "2008" @default.
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