DNA damage repair plays a crucial role in maintaining genomic stability, and mutations in related genes can compromise genomic integrity, increasing the risk of cancer. DNA-damaging agents such as cisplatin and ionizing radiation (IR) are widely used in cancer treatment, but clinical studies have increasingly identified resistance mutations in patients undergoing these therapies.

 

 

On December 11, 2024, Wensheng Wei's team at Peking University published a research paper titled "Mapping functional elements of the DNA damage response through base editor screens" in Cell Reports. Building on their previously developed lysine functional site screening strategy and gene knockout strategies^1-3, they employed adenine base editors and cytosine base editors to target and edit lysine sites or genes across the genome, resulting in lysine site mutations or gene knockouts. They then conducted screenings in the human retinal pigment epithelial cell line (RPE1) and combined these with treatments using two DNA-damaging agents, ultimately identifying several key amino acid sites and genes involved in the DNA damage repair process (Figure 1).

 

Figure 1: Screening workflow and results

In the cisplatin screening results, the top ten negatively ranked genes were mostly known key factors in the FA repair pathway (e.g., FANCM, FANCA, FANCG). The study revealed that the STK35 gene, previously unrelated to DNA damage response, plays a significant role in DNA repair. Literature shows that its family members, STK19 and STK11, are involved in the TC-NER pathway and UVB-induced damage repair, respectively. Subsequent experiments confirmed that STK35 is a potential DNA repair factor, playing a crucial role in the DNA repair process.

 

In 2019, C17orf53 was identified as a key factor in interstrand crosslink (ICL) repair. This study found that the K494 site of C17orf53 is a critical amino acid site for its role in DNA damage repair. Mutation at K494 disrupts the interaction between C17orf53 and its upstream factor RPA, leading to severe ICL repair defects, G2/M cell cycle arrest, and significantly increased sensitivity to cisplatin treatment.

 

Additionally, the study identified several important post-translational modification sites related to DNA damage repair, such as the K120 site of p53, which is a key acetylation site regulating its apoptotic function. Mutation at this site can result in high tolerance to DNA-damaging agents.

 

In summary, this study systematically mapped functional lysine sites and related genes influencing the DNA damage response using various base editing screening strategies. This provides a new perspective for studying the functions of DNA damage response genes and accelerates research into resistance mutations in cancer treatment (Figure 2).

 

Figure 2: Summary diagram

 

The co-first authors of this study are Dr. Qian Pan (graduated) and Zhixuan Zhang from Wei Wensheng's lab at Peking University. Yangfang Xiong, Ying Bao, Tianxin Chen, and Dr. Ping Xu (graduated) also made significant contributions. The research was supported by the National Natural Science Foundation of China, the Peking-Tsinghua Center for Life Sciences, and Changping Laboratory.

 

Article link: https://doi.org/10.1016/j.celrep.2024.115047

 

References:

1. Xu, P., Liu, Z., Liu, Y., Ma, H., Xu, Y., Bao, Y., Zhu, S., Cao, Z., Wu, Z., Zhou, Z., et al. (2021). Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs. Nat Biotechnol 39, 1403--1413. https://doi.org/10.1038/s41587-021-00944-1.

2. Zhu, S., Cao, Z., Liu, Z., He, Y., Wang, Y., Yuan, P., Li, W., Tian, F., Bao, Y., and Wei, W. (2019). Guide RNAs with embedded barcodes boost CRISPR-pooled screens. Genome Biology 20. https://doi.org/10.1186/s13059-019-1628-0.

3. Bao, Y., Pan, Q., Xu, P., Liu, Z., Zhang, Z., Liu, Y., Xu, Y., Yu, Y., Zhou, Z., and Wei, W. (2023). Unbiased interrogation of functional lysine residues in human proteome. Molecular Cell 83, 4614-4632.e6. https://doi.org/10.1016/j.molcel.2023.10.033.