The Intrinsically Disordered Region in the Human STN1 OB-Fold Domain Is Important for Protecting Genome Stability

SIMPLE SUMMARY: The human CTC1–STN1–TEN1 (CST) complex is an ssDNA-binding protein complex that is thought to be related to the RPA70/RPA32/RPA14 complex. While recent studies have shown that CST plays key roles in multiple genome maintenance pathways, including protecting fork stability under perturbed replication, promoting efficient replication of difficult-to-replicate DNA, repairing DNA double-stranded breaks, and maintaining telomere integrity, it is poorly understood how CST function is regulated in genome maintenance. In this study, we identify an intrinsically disordered region (IDR) in the OB domain of STN1 and analyze the functions of cancer-associated IDR variants and a number of alanine substitutions of individual polar or hydrophilic residues in this IDR. We observe that these variants confer replication-associated genome instability, reduced cellular viability, and increased HU sensitivity. Analysis of protein–protein interactions using IDR variants and IDR deletion shows that the IDR is critical for STN1–POLα interaction, but not CST–RAD51 interaction or CST complex formation. Together, our results identify the IDR in STN1-OB as an important element modulating CST function in protecting genome stability under replication stress. ABSTRACT: The mammalian CTC1–STN1–TEN1 (CST) complex is an ssDNA-binding protein complex that has emerged as an important player in protecting genome stability and preserving telomere integrity. Studies have shown that CST localizes at stalled replication forks and is critical for protecting the stability of nascent strand DNA. Recent cryo-EM analysis reveals that CST subunits possess multiple OB-fold domains that can form a decameric supercomplex. While considered to be RPA-like, CST acts distinctly from RPA to protect genome stability. Here, we report that while the OB domain of STN1 shares structural similarity with the OB domain of RPA32, the STN1-OB domain contains an intrinsically disordered region (IDR) that is important for maintaining genome stability under replication stress. Single mutations in multiple positions in this IDR, including cancer-associated mutations, cause genome instabilities that are elevated by replication stress and display reduced cellular viability and increased HU sensitivity. While IDR mutations do not impact CST complex formation or CST interaction with its binding partner RAD51, they diminish RAD51 foci formation when replication is perturbed. Interestingly, the IDR is critical for STN1–POLα interaction. Collectively, our results identify the STN1 IDR as an important element in regulating CST function in genome stability maintenance.