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Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase
L V Mendelman1, J Petruska, M F Goodman
1Department of Biological Sciences, University of Southern California, Los Angeles 90089-1340.
The Journal of Biological Chemistry
|February 5, 1990
Summary
This study compares DNA polymerase alpha and reverse transcriptase in extending mismatched DNA bases. Reverse transcriptase is more efficient with transition mismatches, while polymerase alpha handles some transversion mismatches better, impacting mutation rates.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- DNA polymerases and reverse transcriptases are crucial for DNA replication and repair.
- Understanding their fidelity in extending mismatched base pairs is essential for comprehending mutation processes.
Purpose of the Study:
- To kinetically analyze the extension of single deoxyribonucleotide misincorporations by Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase.
- To compare the extension efficiencies of all possible DNA base pair mismatches for these two enzymes.
Main Methods:
- A polyacrylamide gel assay was employed to measure the kinetics of deoxyribonucleotide addition to primer termini on an M13 template.
- Enzyme kinetics were assessed for all DNA base pair and mispair combinations using purified Pol alpha and reverse transcriptase.
Main Results:
- Reverse transcriptase demonstrated higher efficiency in extending transition mispairs (Pur.Pyr, Pyr.Pur) compared to Pol alpha, notably with G.T.
- For transversion mispairs (Pur.Pur, Pyr.Pyr), enzyme efficiencies varied, with Pol alpha showing higher extension rates for A.G and G.G, while reverse transcriptase was better for C.T and T.T.
- Both enzymes showed very low extension efficiencies for G.G mispairs relative to correct G.C pairing.
- General rules emerged: transition mispairs are easily inserted and extended, Pyr.Pyr mispairs are difficult to insert but slightly easier to extend, and Pur.Pur mispairs are harder to extend than insert.
- Reverse transcriptase generally extends mismatches more efficiently than it forms them, except for G.G.
- Pol alpha efficiently inserts but poorly extends A.A mismatches, potentially reducing in vivo transversion mutations.
- Extension velocities showed positive cooperativity with substrate concentration, deviating from Michaelis-Menten kinetics.
Conclusions:
- The differential extension efficiencies of Pol alpha and reverse transcriptase on various DNA mismatches contribute to their distinct roles in cellular processes and mutation avoidance.
- The findings provide insights into the mechanisms of DNA fidelity and the origins of point mutations.
- The observed deviation from Michaelis-Menten kinetics suggests complex regulatory mechanisms influencing polymerase activity in response to substrate availability.