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The Saccharomyces cerevisiae DBR1 gene encodes a 2'-5' phosphodiesterase that debranches intron RNA lariats following splicing. Yeast dbr1 mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize to ACT1 intron RNA. Protection patterns confirm that sequences from the 5' end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generated dbr1 mutant alleles are also deficient for debranching, further supporting a role for 2'-5' phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently of DBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.  相似文献   

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The Ty5 retrotransposon of Saccharomyces paradoxus transposes in Saccharomyces cerevisiae at frequencies 1,000-fold lower than do the native Ty1 elements. The low transposition activity of Ty5 could be due to differences in cellular environments between these yeast species or to naturally occurring mutations in Ty5. By screening of a Ty5 mutant library, two single mutants (D252N and Y68C) were each found to increase transposition approximately sixfold. When combined, transposition increased 36-fold, implying that the two mutations act independently. Neither mutation affected Ty5 protein synthesis, processing, cDNA recombination, or target site choice. However, cDNA levels in both single mutants and the double mutant were significantly higher than in the wild type. The D252N mutation resides in the zinc finger of nucleocapsid and increases the potential for hydrogen bonding with nucleic acids. We generated other mutations that increase the hydrogen bonding potential (i.e., D252R and D252K) and found that they similarly increased transposition. This suggests that hydrogen bonding within the zinc finger motif is important for cDNA production and builds upon previous studies implicating basic amino acids flanking the zinc finger as important for zinc finger function. Although NCp zinc fingers differ from the zinc finger motifs of cellular enzymes, the requirement for efficient hydrogen bonding is likely universal.  相似文献   

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E. coli RNA polymerase is shown to be capable of catalyzing consecutive DNA-dependent pyrophosphorolysis of RNA in the presence of inorganic pyrophosphate and Mg2+. Active ternary complex of the enzyme with DNA and nascent RNA is needed for the reaction, the mixure of all the components can not carry out pyrophosphorolysis. The reaction was realized in the absence of added nucleoside triphosphates. Nucleoside triphosphates are low molecular mass products of the reaction. The rate of pyrophosphorolysis of the RNA synthesised for the AI promoter of the DNA of wild type T7 phage and delta D III T7 mutant phage was followed as a function of primary structure of the DNA, temperature, ionic strength and inorganic pyrophosphate concentration. The relative rate pyrophosphorolysis for particular nucleotides in different regions of the RNA can differ by several orders of magnitude depending on the primary structure of the RNA that undergoes pyrophosphorolysis. Ternary complex containing partially pyrophosphorilised RNA is active on the RNA synthesis when pyrophosphate is removed and nucleoside triphosphates are added to the reaction mixture. RNA as short as 70-8 nucleotides long can be produced at the conditions used. It seems that efficient dissociation in this region of RNA limits the pyrophosphorolysis to proceed up to the 5' end of RNA. Ternary complex of RNA polymerase with nascent RNA and DNA is shown to undergo site specific dissociation. The specificity of the dissociation is shown to be a function of the primary structure of RNA and the direction of the reaction. Dissociation occurs at different places along RNA sequence when the RNA is synthesised and when it is pyrophosphorilised.  相似文献   

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Singh K  Modak MJ 《Biochemistry》2005,44(22):8101-8110
Previous structural and biochemical data indicate a participation of the J-helix of Escherichia coli pol I in primer positioning at the polymerase and exonuclease sites. The J-helix contains three polar residues: N675, Q677, and N678. Preliminary characterization of alanine substitutions of these residues showed that only Q677A DNA polymerase has substantially decreased polymerase and increased exonuclease activity. The Q677A enzyme had approximately 2- and approximately 5-fold greater exonuclease activity than the wild type (WT) with mismatched and matched template-primers (TPs), respectively. N675A and N678A DNA polymerases did not differ significantly from the WT in these activities, despite the fact that both residues are seen to interact with the TP in various pol I-DNA complexes. Pre-steady-state kinetic measurements for the exonuclease activity of WT and mutant enzymes indicated nearly identical DNA binding affinity for ssDNA and mismatched TPs. However, with a matched TP, Q677A DNA polymerase exhibited increased exonuclease site affinity. The most important characteristic of Q677A DNA polymerase was its ability to continue cleavage into the matched region of the TP after mismatch excision, in contrast to the WT and other mutant enzymes. The increase in the exonuclease activity of Q677A DNA polymerase was further determined not to be solely due to the weakened binding at the polymerase site, by comparison with another polymerase-defective mutant enzyme, namely, R668A DNA polymerase. These enzymes have significantly decreased DNA binding affinity at the polymerase site, yet the exonuclease activity parameters of R668A DNA polymerase remain similar to those of the WT. These results strongly suggest that participation of Q677 is required for positioning the primer terminus (a) in the polymerase site for continued nucleotide addition and (b) in the 3'-exonuclease site for the controlled removal of mismatched nucleotides.  相似文献   

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Vaisman A  Ling H  Woodgate R  Yang W 《The EMBO journal》2005,24(17):2957-2967
We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not.  相似文献   

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The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.  相似文献   

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