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Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase
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...
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2008
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2148107/ https://www.ncbi.nlm.nih.gov/pubmed/18167548 http://dx.doi.org/10.1371/journal.pone.0001389 |
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author | Pandey, Manjula Patel, Smita S. Gabriel, Abram |
author_facet | Pandey, Manjula Patel, Smita S. Gabriel, Abram |
author_sort | Pandey, Manjula |
collection | PubMed |
description | 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 (D(211)) 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 D(211)N mutant enzymes, and study their pre-steady state kinetic parameters. We found that the k(pol) was reduced by a factor of 223 in the mutant, although the K(d) for nucleotide binding was unaltered. Further, the mutant enzyme had a marked preference for Mn(2+) over Mg(2+). 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 D(211)N 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 (k(pyro)) in the D(211)N mutant is nearly 320 fold lower than the WT enzyme, and that the mutant enzyme has an ∼170 fold lower apparent K(d) 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 D(211) residue has a role in pyrophosphate binding and release, which could affect polymerase translocation, and help explain the D(211)N mutant's transposition defect. |
format | Text |
id | pubmed-2148107 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-21481072008-01-02 Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase Pandey, Manjula Patel, Smita S. Gabriel, Abram PLoS One Research Article 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 (D(211)) 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 D(211)N mutant enzymes, and study their pre-steady state kinetic parameters. We found that the k(pol) was reduced by a factor of 223 in the mutant, although the K(d) for nucleotide binding was unaltered. Further, the mutant enzyme had a marked preference for Mn(2+) over Mg(2+). 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 D(211)N 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 (k(pyro)) in the D(211)N mutant is nearly 320 fold lower than the WT enzyme, and that the mutant enzyme has an ∼170 fold lower apparent K(d) 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 D(211) residue has a role in pyrophosphate binding and release, which could affect polymerase translocation, and help explain the D(211)N mutant's transposition defect. Public Library of Science 2008-01-02 /pmc/articles/PMC2148107/ /pubmed/18167548 http://dx.doi.org/10.1371/journal.pone.0001389 Text en Pandey et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Pandey, Manjula Patel, Smita S. Gabriel, Abram Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase |
title | Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase |
title_full | Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase |
title_fullStr | Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase |
title_full_unstemmed | Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase |
title_short | Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase |
title_sort | kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2148107/ https://www.ncbi.nlm.nih.gov/pubmed/18167548 http://dx.doi.org/10.1371/journal.pone.0001389 |
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