RNA transcripts serve as a template for double-strand break repair in human cells

• DOI: 10.1038/s41467-025-59510-x
• Publication Date: 2025-05-10T10:58:39Z
• Authors: Manisha Jalan; Alessandra Brambati; Hina Shah; Niamh McDermott; Juber Patel; Yingjie Zhu; Ahmet Doymaz; Julius Wu; Kyrie S. Anderson; Andrea Gazzo; Fresia Pareja; Takafumi N. Yamag...; Theodore Vougiouk...; Sana Ahmed-Seghir; Philippa Steinberg; Anna Neiman-Golden; Benura Azeroglu; Joan Gomez-Aguilar; Edaise M. da Silva; Suleman Hussain; Daniel Higginson; Paul C. Boutros; Nadeem Riaz; Jorge S. Reis-Filho; Simon N. Powell; Agnel Sfeir

Abstract Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, growing evidence suggests RNA:DNA hybrids and nearby transcripts can influence outcomes. However, whether transcript RNA directly serve as a template for in human cells remains unclear. In this study, we develop fluorescence sequencing-based assays show RNA-containing oligonucleotides messenger templates during repair. We conduct CRISPR/Cas9-based genetic screen identify factors promote RNA-templated (RT-DSBR). Of the candidate polymerases, DNA polymerase zeta (Polζ) potential reverse transcriptase facilitates RT-DSBR. Furthermore, analysis cancer sequencing data reveals whole intron deletions - distinct genomic signature RT-DSBR occurs when spliced mRNA guides Altogether, our findings highlight an alternative pathway repairing DSBs transcribed genes, with mutagenic consequences.