University of Split School of Medicine, Laboratory for cancer research, Split 21000, Croatia.
Mediterranean Institute for Life Sciences (MedILS), Split 21000, Croatia.
Nucleic Acids Res. 2023 Apr 11;51(6):e35. doi: 10.1093/nar/gkad022.
DNA-protein crosslinks (DPCs), formed by the covalent conjugation of proteins to DNA, are toxic lesions that interfere with DNA metabolic processing and transcription. The development of an accurate biochemical assay for DPC isolation is a priority for the mechanistic understanding of their repair. Here, we propose the STAR assay for the direct quantification of DPCs, sensitive to physiologically relevant treatment conditions. Implementing the STAR assay revealed the formation of small cross-linked peptides on DNA, created by the proteolytic degradation of DPCs by SPRTN. The initial proteolytic degradation of DPCs is required for the downstream activation of DNA repair, which is mediated through the phosphorylation of H2Ax. This leads to the accumulation of DNA repair factors on chromatin and the subsequent complete removal of the cross-linked peptides. These results confirmed that the repair of DPCs is a two-step process, starting with proteolytic resection by SPRTN, followed by the repair of the underlying damage to the DNA.
DNA-蛋白质交联（DPCs）是指蛋白质与 DNA 发生共价连接形成的毒性损伤，会干扰 DNA 代谢过程和转录。因此，开发一种准确的生化方法来分离 DPCs 对于深入了解其修复机制至关重要。本文提出了 STAR 检测法，可直接定量分析 DPCs，且对生理相关的处理条件具有较高的灵敏度。该检测法揭示了 SPRTN 通过蛋白水解作用切割 DPCs 产生的小交联肽在 DNA 上的形成。DPCs 的初始蛋白水解切割对于下游的 DNA 修复激活是必需的，这一过程是通过 H2Ax 的磷酸化来介导的。这会导致 DNA 修复因子在染色质上的积累，并随后完全去除交联肽。这些结果证实，DPCs 的修复是一个两步过程，首先由 SPRTN 进行蛋白水解切除，然后修复 DNA 上的潜在损伤。
