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Related Concept Videos

Nucleotide Excision Repair01:08

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The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
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DNAzyme-dependent Analysis of rRNA 2’-O-Methylation
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Sequence and functional-group specificity for cleavage of DNA junctions by RuvC of Escherichia coli.

J M Fogg1, M J Schofield, M F White

  • 1CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University of Dundee, United Kingdom.

Biochemistry
|September 2, 1999
PubMed
Summary

The RuvC enzyme resolves DNA junctions, showing sequence preference for cleavage. Optimal cleavage occurs at specific DNA sequences, particularly TT, influencing the enzyme's transition state and DNA conformation.

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Area of Science:

  • Molecular Biology
  • Enzymology
  • DNA Repair Mechanisms

Background:

  • RuvC is the DNA junction-resolving enzyme in Escherichia coli.
  • While RuvC binds DNA junctions non-specifically, it displays sequence selectivity during phosphodiester bond cleavage.

Purpose of the Study:

  • To analyze the sequence specificity of RuvC for DNA cleavage.
  • To determine the optimal DNA sequence for RuvC-mediated phosphodiester bond scission.

Main Methods:

  • Assessed RuvC sequence specificity using a panel of DNA junctions.
  • Measured cleavage rates under single-turnover conditions.
  • Utilized base analogues to probe functional group importance.

Main Results:

  • Identified an optimal cleavage sequence: (A approximately T)TT↓(C>G approximately A), with cleavage fastest at the strand exchange point.
  • Sequence alterations in the central TT motif caused at least a 100-fold rate reduction.
  • Major-groove contacts within the tetranucleotide site are crucial for cleavage, impacting the transition state.

Conclusions:

  • RuvC exhibits significant sequence selectivity for DNA cleavage, primarily dictated by the central TT motif.
  • The enzyme's sequence preference suggests critical transition-state interactions, likely involving major-groove contacts.
  • Optimal sequences enhance RuvC cleavage efficiency on both four-way and three-way DNA junctions.