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Updated: May 23, 2025
Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells
Published on: September 8, 2010
RNA Transcripts Serve as a Template for Double-Strand Break Repair in Human Cells
Abstract:
Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, emerging evidence suggests that RNA:DNA hybrids and transcripts near damaged sites can influence repair outcomes. However, a direct role for transcript RNA as a template during DSB repair in human cells is yet to be established. In this study, we designed fluorescent- and sequencing-based assays, which demonstrated that RNA-containing oligonucleotides and messenger RNA serve as templates to promote DSB repair. We conducted a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR), and of the candidate polymerases, we identified DNA polymerase-zeta (Polζ) as the potential reverse transcriptase that facilitates RT-DSBR. Furthermore, by analyzing sequencing data from cancer genomes, we identified the presence of whole intron deletions, a unique genomic scar reflective of RT-DSBR activity generated when spliced mRNA serves as the repair template. These findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.
Insights
This study reveals that RNA can template DNA repair in human cells, a process called RNA-templated DNA repair (RT-DSBR). DNA polymerase-zeta (Polζ) facilitates this pathway, which can leave unique genomic scars like intron deletions.
Area of Science:
- Molecular Biology
- Genetics
- Genomics
Background:
- Double-strand breaks (DSBs) are critical DNA lesions that compromise genome stability.
- Canonical DNA repair pathways typically do not involve RNA, but recent findings suggest RNA's potential influence on repair outcomes.
- The direct role of transcript RNA as a template in human cellular DSB repair remains largely uncharacterized.
Purpose of the Study:
- To investigate the direct role of RNA as a template in double-strand break repair in human cells.
- To identify factors and mechanisms governing RNA-templated DNA repair (RT-DSBR).
- To explore the genomic consequences of RT-DSBR in cancer.
Main Methods:
- Development of fluorescent and sequencing-based assays to detect RNA-templated repair.
- CRISPR/Cas9-based genetic screening to identify key factors in RT-DSBR.
- Bioinformatic analysis of cancer genome sequencing data.
Main Results:
- Demonstrated that RNA oligonucleotides and messenger RNA can serve as templates to promote DSB repair.
- Identified DNA polymerase-zeta (Polζ) as a key enzyme, potentially acting as a reverse transcriptase, that facilitates RT-DSBR.
- Discovered whole intron deletions in cancer genomes as a distinct genomic scar indicative of RT-DSBR using spliced mRNA.
Conclusions:
- RNA-templated DNA repair (RT-DSBR) is an alternative pathway for repairing DSBs within transcribed genes.
- RT-DSBR involves Polζ and can lead to specific mutagenic outcomes, such as intron deletions.
- The findings reveal a novel mechanism of DNA repair with implications for genome stability and cancer mutagenesis.
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