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Updated: Jun 9, 2025
07:38
DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
Published on: October 6, 2017
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DNA polymerase ζ has robust reverse transcriptase activity relative to other cellular DNA polymerases
Ryan Mayle1, William K Holloman2, Michael E O'Donnell1
1Howard Hughes Medical Institute and the Department of DNA Replication, The Rockefeller University, New York, New York, USA.
The Journal of Biological Chemistry
|October 25, 2024
Summary
Yeast Pol ζ is the primary enzyme for RNA-templated DNA double-strand break (DSB) repair. This study found Pol ζ exhibits superior reverse transcriptase activity compared to other DNA polymerases (Pol), facilitating RNA-guided DNA repair.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA double-strand breaks (DSBs) can be repaired using RNA templates.
- This process requires reverse transcriptase activity to synthesize DNA from an RNA template.
- Translesion synthesis (TLS) DNA polymerases (Pol) are candidates due to their promiscuity.
Purpose of the Study:
- To compare the reverse transcriptase activities of all seven known nuclear DNA polymerases in Saccharomyces cerevisiae.
- To identify the primary DNA polymerase involved in RNA-templated DSB repair.
Main Methods:
- Purification of all seven nuclear DNA polymerases from Saccharomyces cerevisiae.
- Assay of reverse transcriptase activity for each purified polymerase.
- Analysis of Pol ζ activity in the presence of RPA and RFC/PCNA.
Main Results:
- Yeast Pol ζ demonstrated significantly higher reverse transcriptase activity than human Pol η and other DNA polymerases.
- Pol ζ's reverse transcriptase activity was independent of RPA and RFC/PCNA.
- Pol ζ synthesized DNA complementary to an RNA template in a distributive manner.
Conclusions:
- Pol ζ is the major DNA polymerase functioning in RNA-templated DSB repair in Saccharomyces cerevisiae.
- These findings support the role of TLS polymerases in non-canonical DNA repair pathways.
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