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Kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase.

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 (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 approximately 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.

SUBMITTER: Pandey M

PROVIDER: S-EPMC2148107 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase.

Pandey Manjula M Patel Smita S SS Gabriel Abram A

PloS one 20080102 1

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 supp ...[more]

PMID: 18167548

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Project description:LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells.

Dataset Information

Kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase.

Publications

Kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase.

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