Cells are frequently subjected to DNA damage from endogenous and exogenous sources (Awwad et al., 2023, Lord and Ashworth, 2012). Among the various types of DNA damage, DNA double-strand breaks (DSBs) are considered the most damaging form (Huang and Zhou, 2021, Roos et al., 2016). The accumulation of such damage can result in high mutation rates, activation of oncogenes, and inactivation of tumor suppressor genes, ultimately leading to genomic instability and tumorigenesis (Basu, 2018, Huang and Zhou, 2021, Negrini et al., 2010, Roos et al., 2016). Conventional cancer therapies, such as radiotherapy and chemotherapy, target tumor cells by inducing various types of DNA damage, particularly DSBs (Geng et al., 2024; O′Connor, 2015; Pilié et al., 2019). Therefore, the ability of cells to repair DNA damage promptly and effectively has a crucial role in the initiation and progression of tumors, as well as in determining the response of tumor cells to treatment. Investigating the mechanisms underlying DNA repair in depth holds significant clinical importance in reducing tumor occurrence and guiding personalized treatment approaches for cancer patients.

DSBs represent the most cytotoxic form of DNA damage and their repair is crucial for maintaining genome stability (Huang and Zhou, 2021, Roos et al., 2016). Cells primarily repair DSBs through non-homologous end joining (NHEJ) and homologous recombination (HR) (Chen et al., 2019, Geng et al., 2024, Liu et al., 2018b, Mao et al., 2011). NHEJ repair involves DNA ligase directly connecting two broken DSB ends and is active in all cell cycles (Mao et al., 2008). In contrast, HR repair is a multi-step complex process that maintains genome integrity without causing nucleotide mutations or deletions (Mao et al., 2008). HR mainly occurs during the S and G2 phases of the cell cycle and need sister chromatids as repair templates (Chen et al., 2019, Mao et al., 2008). Despite the significance of NHEJ and HR repair, comprehensive understanding of repair processes at the molecular level is lacking. Recent studies have shed light on the role of RNA in DNA damage repair, particularly the presence of damage-induced long non-coding RNA (dilncRNA) and small DNA-damage response (DDR) RNA (DDRNA) at DSB sites (Francia et al., 2012, Liu et al., 2021, Liu et al., 2022b, Michelini et al., 2018, Michelini et al., 2017, Petermann et al., 2022). While the mechanisms underlying RNA biogenesis have not been fully elucidated, emerging evidence suggests that dilncRNAs form DNA:RNA hybrids at DSBs, which facilitates recruitment of DNA repair proteins (D'Alessandro et al., 2018, Francia et al., 2016, Liu et al., 2018a, Liu et al., 2018c, Lu et al., 2018). However, the mechanisms underlying the regulation and formation of these RNAs at DNA damage sites, as well as the contribution to promoting DNA repair, have not been established.

RNA modification has a crucial role in RNA metabolism, encompassing > 170 identified types of modifications, including N6-methyladenosine (m6A), N4-acetylcytidine (ac4C), 5-methylcytosine (m5C), N7-methylguanosine (m7G), and N1-methyladenosine (m1A) (Roundtree et al., 2017). Several studies, including studies from our own group, have demonstrated that m6A modification affects RNA stability, translation, shearing, and translocation, and has a role in the pathogenesis of multiple tumors (Barbieri and Kouzarides, 2020, Deng et al., 2023, Wang et al., 2020b, Wang et al., 2022, Xia et al., 2022). Recent studies have also shown that m6A modification facilitates ultraviolet (UV)-induced single-strand breaks (SSBs) and recruits DNA polymerase κ (Polκ) to aid in nucleotide excision and trans-lesion synthesis-mediated SSB repair (Xiang et al., 2017b). Additionally, ataxia-telangiectasis mutated (ATM)-phosphorylated N6-adenosine-methyltransferase 70 kDa subunit (METTL3) is recruited to the DSBs, while YTH domain-containing protein 1 (YTHDC1) recognizes and protects the m6A modification of nascent RNA (Zhang et al., 2020). The m6A modification recruits DNA damage repair-related proteins, forming DNA:RNA hybrids at DSBs to regulate HR repair (Zhang et al., 2020). Recent findings have indicated that m5C RNA modification undergoes demethylation during DNA damage repair, with fragile X mental retardation protein (FMRP) interacting with tRNA aspartic acid methyltransferase 1 (TRDMT1) and recruitment to DNA damage sites to assist in ten-eleven translocation methylcytosine dioxygenase 1 (TET1)-mediated demethylation. The absence of FMRP can lead to the accumulation of m5C and related R-loops at DSB sites, which impedes the HR process and increases sensitivity to radiotherapy (Yang et al., 2022). RNA modification has been widely involved in DNA repair, whether ac4C directly regulate DSB repair remains uncertain.

N-acetyltransferase 10 (NAT10) was the sole RNA acetyltransferase mediating RNA ac4C modification, a post-transcriptional mechanism that enhances mRNA stability and translational efficiency (Ge et al., 2024, Wang et al., 2025). NAT10 exhibits significant oncogenic properties, with documented overexpression across multiple malignancies. DNA damage triggers NAT10 upregulation through transcriptional activation, establishing its critical role in DNA damage response and chemoresistance (Liu et al., 2007). DNA damage induces NAT10 nucleoplasmic translocation, enabling its enzymatic acetylation and stabilization of p53 and PARP1 to orchestrate DNA repair machinery (Liu et al., 2022a, Liu et al., 2016, Zhang and Li, 2019). Pharmacological inhibition or genetic depletion of NAT10 significantly sensitizes cancer cells to DNA-damaging agents and radiation by compromising DNA repair mechanisms (Liu et al., 2020). Notably, NAT10 deficiency induces defective DSB repair in germline cells and promotes cisplatin sensitivity in bladder cancer through decreased DNA repair (Chen et al., 2022, Jiang et al., 2023, Xie et al., 2023). Although previous studies established NAT10's functional importance in genomic maintenance, critical questions remain regarding its direct recruitment dynamics to DNA lesions.

This study presents novel findings that show NAT10 facilitates ac4C modification on DNA:RNA hybrids at DSB sites to enhance HR-mediated DSB repair. NAT10 is rapidly recruited to DSB sites upon DSB occurrence by the DNA damage sensor, poly (ADP-ribose) polymerase 1 (PARP1), which leads to RNA ac4C modification on DNA:RNA hybrids that bolster the stability of these hybrids, which in turn, promotes HR repair. In the current study, the previously unexplored role of ac4C modification in DNA damage repair has been clarified. Clinically, NAT10 was upregulated in hepatocellular carcinoma (HCC) and ablation of NAT10 in murine hepatocytes suppressed HCC progression. Furthermore, high-resolution structures of NAT10 in complex with the NAT10 inhibitor, remodelin, have been acquired through cryo-electron microscopy (cryo-EM). This structure yielded a remarkable 2.9-angstrom resolution structure, showcasing a C2 symmetric architecture. Remodelin treatment significantly enhanced the sensitivity of HCC cells to PARP inhibitors (PARPis). Importantly, targeting NAT10 in clinical translational research also restored sensitivity to PARPis in ovarian and breast cancer cells that developed resistance to PARPis. These finding underscore an NAT10 mechanism in HR repair that was previously not described and point towards a promising therapeutic strategy combining remodelin and a PARPi for treatment of PARPi-resistant cancers.