Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacterial reverse transcriptase (RT) is responsible for synthesis of multicopy single-stranded DNA (msDNA) consisting of single-stranded DNA linked to an internal guanosine residue of RNA by an unusual 2',5'-phosphodiester linkage. Here we purified a bacterial RT to homogeneity from Escherichia coli harboring the RT gene from retron-Ec73. The purified RT-Ec73 was able to synthesize msDNA in a cell-free system using an RNA template produced in vitro by T7 RNA polymerase. The in vitro synthesized msDNA was released from the template RNA only when treated with yeast debranching enzyme DBR1, a specific nuclease for a 2',5'-phosphodiester linkage. The position of the branching G residue in the template RNA and the DNA sequence of the cell-free product were identical to those of msDNA-Ec73 synthesized in vivo. These results clearly demonstrate that the formation of the 2',5'-phosphodiester linkage in msDNA synthesis is carried out by RT itself.
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PMID:The formation of the 2',5'-phosphodiester linkage in the cDNA priming reaction by bacterial reverse transcriptase in a cell-free system. 752 62

A mutant screen has been initiated to identify host genes important for the replication of retrotransposons in Saccharomyces cerevisiae. Two mutants were identified that undergo Ty1 and Ty3 transposition at <10% of the wild-type frequency. Both these mutants have deficiencies in the accumulation of full-length Ty1 and Ty3 cDNAs, although Ty proteins (including reverse transcriptase) accumulate at wild-type levels. The DBR1 gene, encoding the yeast debranching enzyme, complements both mutants. This suggests that Dbr1p is important for either reverse transcription or the stability of Ty cDNA, roles that have not been previously reported for this protein. The deficiency in accumulation of Ty cDNAs in dbr1 mutants is apparent when engineered Ty elements are expressed for short time periods (6-10 h) but is not apparent following long expression periods (>24 h).
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PMID:The yeast retrotransposons Ty1 and Ty3 require the RNA Lariat debranching enzyme, Dbr1p, for efficient accumulation of reverse transcripts. 1065 22

2',5'-branched RNA was recently proposed as a key Ty1 retrotransposition intermediate, for which cleavage by lariat debranching enzyme (Dbr1p) enables reverse transcription to continue synthesizing the complete Ty1 cDNA. Because dbr1 cells can produce substantial Ty1 cDNA despite lacking Dbr1p, the obligatory intermediacy of branched RNA would require that Ty1 reverse transcriptase (RT) can read through the proposed branch site with considerable efficiency. Here we have used deoxyribozyme-synthesized 2',5'-branched RNA corresponding exactly to the proposed Ty1 branch site for a direct test of this read-through ability. Using an in vitro assay that incorporates all components known to be required for Ty1 cDNA synthesis (including the TyA chaperone protein), Ty1 RT can elongate up to the branch site. Strand transfer from the 2'-arm to the 3'-arm of the branch is observed when the Ty1 RT is RNase H+ (i.e., wild-type) but not when the Ty1 RT is RNase H-. When elongating from either the 2'-arm or the 3'-arm, Ty1 RT reads through the branch site with <or=0.3% efficiency. This is at least 60-fold lower than would be necessary to explain in vivo Ty1 cDNA synthesis in dbr1 cells, because others have reported 18% cDNA synthesis relative to wild-type cells. Our finding that Ty1 RT cannot efficiently read through the proposed Ty1 branch site is inconsistent with the hypothesis that branched RNA is an obligatory Ty1 retrotransposition intermediate. This suggests that Dbr1p acts as other than a 2',5'-phosphodiesterase during Ty1 retrotransposition.
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PMID:Ty1 reverse transcriptase does not read through the proposed 2',5'-branched retrotransposition intermediate in vitro. 1765 36

Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5' cDNA ends (5' RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it.IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.
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PMID:Conformational Changes in the 5' End of the HIV-1 Genome Dependent on the Debranching Enzyme DBR1 during Early Stages of Infection. 2893 90