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Conferring RNA polymerase activity to a DNA polymerase: a single residue in reverse transcriptase controls substrate
G Gao1, M Orlova, M M Georgiadis
1Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Summary
Researchers identified a single amino acid in DNA polymerases that dictates their substrate preference. Mutating this residue allowed the DNA polymerase to act as an RNA polymerase, suggesting a common evolutionary origin for both enzyme types.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Traditional classification of nucleic acid polymerases distinguishes DNA and RNA polymerases based on nucleotide incorporation preference.
- Moloney murine leukemia virus reverse transcriptase is a strict DNA polymerase.
Purpose of the Study:
- To investigate the role of a specific active site residue in discriminating against ribonucleotides.
- To determine if altering this residue could change the polymerase's substrate specificity.
Main Methods:
- Structure determination of Moloney murine leukemia virus reverse transcriptase.
- Site-directed mutagenesis of a key active site residue.
- Enzyme kinetics assays comparing wild-type and mutant enzymes with deoxyribonucleotides and ribonucleotides.
Main Results:
- A single amino acid residue was predicted to be responsible for discriminating against the 2'-hydroxyl group of ribonucleotides.
- Mutation of this residue created a variant enzyme capable of acting as an RNA polymerase.
- The mutant enzyme exhibited comparable K(m) values for both ribonucleotides and deoxyribonucleotides, unlike the wild-type enzyme.
Conclusions:
- A single active site residue governs the substrate specificity of nucleic acid polymerases.
- These findings support a common evolutionary origin and a shared catalytic mechanism for DNA and RNA polymerases.
- The study provides insights into the flexibility and evolutionary adaptability of polymerases.