Wootae Kim^1,2,6, Fei Zhao^1,2,6, Rentian Wu^1,2, Sisi Qin¹, Somaira Nowsheen^1,2,3, Jinzhou Huang^1,2, Qin Zhou^1,2, Yuping Chen^4,5, Min Deng^1,2, Guijie Guo^1,2, Kuntian Luo^1,2, Zhenkun Lou^1,2,* & Jian Yuan^4,5,*

¹ Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
² Department of Oncology, Mayo Clinic, Rochester, MN 55905, USA.
³ Mayo Clinic Medical Scientist Training Program, Mayo Clinic Alix School Of Medicine and Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, MN 55905, USA.
⁴ Research Center for Translational Medicine, East Hospital, Tongji University School of Medicine, No. 150 Jimo Road, Shanghai 200120, China.
⁵ Key Laboratory of Arrhythmia, Ministry of Education, East Hospital, Tongji University School of Medicine, No. 150 Jimo Road, Shanghai 200120, China.
⁶ These authors contribute equally: Wootae Kim

^*Correspondence to Zhenkun Lou or Jian Yuan

DNA replication stress-mediated activation of the ATR kinase pathway is important for maintaining genomic stability. In this study, we identified a zinc finger protein, ZFP161 that functions as a replication stress response factor in ATR activation. Mechanistically, ZFP161 acts as a scaffolding protein to facilitate the interaction between RPA and ATR/ATRIP. ZFP161 binds to RPA and ATR/ATRIP through distinct regions and stabilizes the RPA–ATR–ATRIP complex at stalled replication forks. This function of ZFP161 is important to the ATR signaling cascade and genome stability maintenance. In addition, ZFP161 knockout mice showed a defect in ATR activation and genomic instability. Furthermore, low expression of ZFP161 is associated with higher cancer risk and chromosomal instability. Overall, these findings suggest that ZFP161 coordinates ATR/Chk1 pathway activation and helps maintain genomic stability.