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Updated: Aug 30, 2026
Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
Published on: June 19, 2018
Lesion bypass by human DNA polymerase mu reveals a template-dependent, sequence-independent nucleotidyl transferase
Shay Covo1, Luis Blanco, Zvi Livneh
1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Abstract:
DNA polymerase mu (pol mu), which is related to terminal deoxynucleotidyl transferase and DNA polymerase beta, is thought to be involved in non-homologous end joining and V(D)J recombination. Pol mu is induced by ionizing radiation and exhibits low fidelity. Analysis of translesion replication by purified human pol mu revealed that it bypasses a synthetic abasic site with high efficiency, using primarily a misalignment mechanism. It can also replicate across two tandem abasic sites, using the same mechanism. Pol mu extends primers whose 3'-terminal nucleotides are located opposite the abasic site. Most remarkably, this extension occurs via a mode of nucleotidyl transferase activity, which does not depend on the sequence of the template. This is not due to simple terminal nucleotidyl transferase activity, because pol mu is unable to add dNTPs to an oligo(dT)29 primer or to a blunt end duplex oligonucleotide under standard conditions. Thus, pol mu is a dual mode DNA-synthesizing enzyme, which can act as either a classical DNA polymerase or as a non-canonical, template-dependent, but sequence-independent nucleotidyl transferase. To our knowledge, this is the first report on a DNA-synthesizing enzyme with such properties. These activities may be required for its function in non-homologous end joining in the processing of DNA ends prior to ligation.
Insights
DNA polymerase mu (pol mu) acts as both a DNA polymerase and a unique template-independent nucleotidyl transferase. This dual activity is crucial for its role in DNA repair pathways like non-homologous end joining.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Enzymology
Background:
- DNA polymerase mu (pol mu) is related to terminal deoxynucleotidyl transferase and DNA polymerase beta.
- Pol mu is implicated in non-homologous end joining and V(D)J recombination.
- It is induced by ionizing radiation and exhibits low fidelity.
Purpose of the Study:
- To investigate the translesion replication capabilities of purified human pol mu.
- To elucidate the mechanism by which pol mu bypasses abasic sites.
- To characterize the unique enzymatic activities of pol mu.
Main Methods:
- Purified human DNA polymerase mu was used for in vitro assays.
- Translesion replication across synthetic abasic sites was analyzed.
- Nucleotidyl transferase activity was assessed under various conditions.
Main Results:
- Pol mu efficiently bypasses single and tandem abasic sites via a misalignment mechanism.
- Pol mu exhibits template-independent, sequence-independent nucleotidyl transferase activity.
- This non-canonical activity is distinct from simple terminal nucleotidyl transferase activity.
Conclusions:
- DNA polymerase mu possesses dual catalytic modes: classical DNA polymerase and non-canonical nucleotidyl transferase.
- These unique enzymatic properties are likely essential for its function in DNA end processing during non-homologous end joining.
- This is the first report of a DNA-synthesizing enzyme with such dual functional capabilities.
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