Jalan Manisha, Brambati Alessandra, Shah Hina, McDermott Niamh, Patel Juber, Zhu Yingjie, Doymaz Ahmet, Wu Julius, Anderson Kyrie S, Gazzo Andrea, Pareja Fresia, Yamaguchi Takafumi N, Vougiouklakis Theodore, Ahmed-Seghir Sana, Steinberg Philippa, Neiman-Golden Anna, Azeroglu Benura, Gomez-Aguilar Joan, da Silva Edaise M, Hussain Suleman, Higginson Daniel, Boutros Paul C, Riaz Nadeem, Reis-Filho Jorge S, Powell Simon N, Sfeir Agnel
Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Nat Commun. 2025 May 10;16(1):4349. doi: 10.1038/s41467-025-59510-x.
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 that RNA:DNA hybrids and nearby transcripts can influence repair outcomes. However, whether transcript RNA can directly serve as a template for DSB repair in human cells remains unclear. In this study, we develop fluorescence and sequencing-based assays to show that RNA-containing oligonucleotides and messenger RNA can serve as templates during DSB repair. We conduct a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR). Of the candidate polymerases, we identify DNA polymerase zeta (Polζ) as a potential reverse transcriptase that facilitates RT-DSBR. Furthermore, analysis of cancer genome sequencing data reveals whole intron deletions - a distinct genomic signature of RT-DSBR that occurs when spliced mRNA guides repair. Altogether, our findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.
双链断裂（DSBs）是导致基因组不稳定的毒性损伤。虽然经典的DSB修复途径通常独立于RNA发挥作用，但越来越多的证据表明，RNA:DNA杂交体和附近的转录本会影响修复结果。然而，转录RNA是否能直接作为人类细胞中DSB修复的模板仍不清楚。在本研究中，我们开发了基于荧光和测序的检测方法，以表明含RNA的寡核苷酸和信使RNA在DSB修复过程中可作为模板。我们进行了基于CRISPR/Cas9的遗传筛选，以鉴定促进RNA模板化DSB修复（RT-DSBR）的因子。在候选聚合酶中，我们鉴定出DNA聚合酶ζ（Polζ）是一种促进RT-DSBR的潜在逆转录酶。此外，对癌症基因组测序数据的分析揭示了全内含子缺失——这是一种独特的RT-DSBR基因组特征，当剪接的mRNA指导修复时会出现。总之，我们的研究结果突出了RT-DSBR作为转录基因中DSB修复的一种替代途径，具有潜在的诱变后果。
