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Updated: Aug 19, 2026
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
Mechanism of gene repair open for discussion
Olga Igoucheva1, Vitali Alexeev, Kyonggeun Yoon
1Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Jefferson Medical College, Philadelphia, PA 19107, USA.
Abstract:
During the last decade, chimeric RNA-DNA oligonucleotides (RDOs) and single-stranded oligodeoxynucleotides have been used to make permanent and specific sequence changes in the genome, with the ultimate goal of curing human genetic disorders caused by mutations. There have been large variations observed in the rate of gene repair in these studies. This has been due, at least in part, to the lack of standardized assay conditions and the paucity of mechanistic studies in the early developmental stages. Previously, it was proposed that strand pairing is the rate-limiting step and mismatch DNA repair is involved in the gene repair process. We propose an alternative model, in which an oligonucleotide is assimilated to the target DNA during active transcription, leading to formation of a transient D-loop. The trafficking of RNA polymerase is interrupted by the D-loop, and the stalled RNA polymerase complex may signal for recruitment of DNA repair proteins, including transcription-coupled DNA repair and nucleotide-excision repair. Thus, oligonucleotides can be considered as a class of DNA-damaging agents that cause a transient but major structural change in DNA. Understanding of the recognition and repair pathways to process this unusual DNA structure may have relevance in physiologic processes, transcription, and DNA replication.
Insights
Chimeric RNA-DNA oligonucleotides (RDOs) can repair genes by forming a D-loop during transcription, stalling RNA polymerase. This process recruits DNA repair proteins, offering new insights into gene editing and genetic disorder treatments.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Chimeric RNA-DNA oligonucleotides (RDOs) and single-stranded oligodeoxynucleotides are used for genome editing to treat genetic disorders.
- Observed variations in gene repair rates are attributed to non-standardized assays and limited mechanistic studies.
Purpose of the Study:
- To propose an alternative model for oligonucleotide-mediated gene repair.
- To investigate the role of transcription and DNA repair pathways in the gene repair process.
Main Methods:
- Proposed a model involving oligonucleotide assimilation into target DNA during active transcription.
- Hypothesized the formation of a transient D-loop interrupting RNA polymerase trafficking.
- Suggested the recruitment of DNA repair proteins, including transcription-coupled and nucleotide-excision repair.
Main Results:
- Oligonucleotides assimilate into target DNA during transcription, forming a D-loop.
- The D-loop stalls RNA polymerase, signaling for DNA repair protein recruitment.
- Oligonucleotides act as DNA-damaging agents, inducing transient structural changes.
Conclusions:
- The proposed model offers a new perspective on gene repair mechanisms mediated by oligonucleotides.
- Understanding these repair pathways is crucial for advancing gene therapy and understanding DNA replication and transcription.
- Oligonucleotides can be viewed as agents that induce specific DNA structural changes, engaging cellular repair machinery.
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