Related Experiment Video
Updated: Nov 17, 2025
Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging
Published on: April 28, 2021
RETRACTED: Human DNA polymerase θ harbors DNA end-trimming activity critical for DNA repair
Karl E Zahn1, Ryan B Jensen2, Richard D Wood3
1Department of Microbiology and Molecular Genetics, University of Vermont, 89 Beaumont Ave., Burlington, VT 05405, USA; Department of Therapeutic Radiology, Yale University, New Haven, CT 06510, USA.
Abstract:
Cancers with hereditary defects in homologous recombination rely on DNA polymerase θ (pol θ) for repair of DNA double-strand breaks. During end joining, pol θ aligns microhomology tracts internal to 5'-resected broken ends. An unidentified nuclease trims the 3' ends before synthesis can occur. Here we report that a nuclease activity, which differs from the proofreading activity often associated with DNA polymerases, is intrinsic to the polymerase domain of pol θ. Like the DNA synthesis activity, the nuclease activity requires conserved metal-binding residues, metal ions, and dNTPs and is inhibited by ddNTPs or chain-terminated DNA. Our data indicate that pol θ repurposes metal ions in the polymerase active site for endonucleolytic cleavage and that the polymerase-active and end-trimming conformations of the enzyme are distinct. We reveal a nimble strategy of substrate processing that allows pol θ to trim or extend DNA depending on the DNA repair context.
Insights
DNA polymerase theta (pol θ) has a dual role in DNA repair. It trims DNA ends before synthesis and aligns microhomology for efficient double-strand break repair in cancers.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Homologous recombination defects in cancers necessitate alternative DNA repair pathways.
- DNA polymerase theta (pol θ) is crucial for repairing DNA double-strand breaks via end joining.
Purpose of the Study:
- To identify the nuclease responsible for trimming 3' ends during pol θ-mediated end joining.
- To characterize the enzymatic activities of DNA polymerase theta.
Main Methods:
- Biochemical assays to assess nuclease and polymerase activities of pol θ.
- Investigating the role of metal ions and nucleotides in enzymatic functions.
- Analyzing distinct enzyme conformations for DNA synthesis and trimming.
Main Results:
- A novel, intrinsic nuclease activity resides within the polymerase domain of pol θ.
- This nuclease activity, distinct from proofreading, requires metal ions and dNTPs.
- Pol θ utilizes metal ions in its active site for both DNA synthesis and endonucleolytic cleavage.
Conclusions:
- Pol θ possesses a dual function, acting as both a polymerase and an endonuclease.
- The enzyme exhibits distinct conformations for DNA trimming and extension.
- This adaptability allows pol θ to effectively manage DNA repair in various contexts.
Related Concept Videos
02:31Homologous Recombination
02:10Translesion DNA Polymerases
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
01:31Proofreading
Errors During Replication are Corrected by the DNA Polymerase...
01:43Proofreading
01:54Base Excision Repair
The first step of...
01:54Base Excision Repair