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

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.

Keywords: CST; STN1; genome instability; replication stress.

Figure 1

STN1 IDR position and conservation among different species. (A) Annotated protein sequences and secondary structures of the N-termini of RPA32 and STN1 containing the OB domains. α-helices are yellow. β-strands are blue. Residues in IDRs between β3/β4 of RPA32-OB and STN1-OB are red. Dots above the sequence indicate residue positions at an interval of 10. (B) Crystal structure of STN1-OB domain and TEN1 and overlay of STN1-OB/TEN1 with RPA32-OB/RPA14. Structures are derived from Protein Data Bank (PDB) with structure code 4JOI. RPA structures are derived from structure code 2PI2 in PDB. (C) Sequence of STN1 IDR in different species. Residues analyzed in this study (T94, E95, S96, S98, S111) are highlighted.

Figure 2

The IDR is important for maintaining chromosome stability. (A) Stable expression of RNAi-resistant (r-STN1) cancer-associated variants Flag-E95G and Flag-S96V. Endogenous STN1 was depleted with shRNA. Full Western blot images are provided in Supplementary Materials. (B) Representative images of chromosome aberrations in STN1 knockdown cells concurrently expressing WT-STN1 or IDR variants. (C,E) Percent of metaphase spreads containing chromosome aberrations. Statistical analysis was performed with one-way ANOVA. N: the number of metaphase spreads analyzed in each sample. Error bars: SEM. ** p < 0.01, * p < 0.05. (D) Stable expression of RNAi-resistant Flag-T94A, Flag-S98A, and Flag-S111A with concurrent depletion of endogenous STN1.

Figure 3

STN1 IDR variants impair cell proliferation. Colony formation results were from 2 independent experiments with triplicate in each experiment. Each value is normalized to the mean of untreated control knockdown. Error bars: SEM. One-way ANOVA was performed by comparing to shSTN1 to calculate statistical significance. **** p < 0.0001, * p < 0.05.

Figure 4

STN1 IDR mutations impair HU-induced RAD51 foci formation. (A) Representative images of RAD51 foci formation from HeLa cells stably expressing RNAi-resistant WT and IDR variants. Endogenous STN1 was concurrently depleted with shRNA. (B) Percent of RAD51 foci positive cells. Results were means from four independent experiments. At least 150 cells were analyzed for each sample. Error bars: SEM. One-way ANOVA with post hoc Tukey was performed by comparing to shSTN1 to calculate statistical significance. *** p < 0.001, ** p < 0.01.

Figure 5

Impact of STN1 IDR mutations on CST complex formation, CST interaction with RAD51 and POLα. (A) Co-IP of STN1 IDR variants with CTC1, TEN1, and RAD51. HEK293T cells were co-transfected with Flag-CTC1, Myc-STN1, and HA-TEN1 and treated with HU (2 mM, 16 h), and co-IP was performed with anti-Myc antibody. Three independent co-IP experiments were performed for each mutant to ensure reproducibility. Representative results are shown. (B) Co-IP of STN1 IDR variants with POLA1. HEK293T cells were co-transfected with Flag-CTC1, Myc-STN1, and HA-TEN1 and treated with or without HU (2 mM, 24 h), and co-IP was performed with anti-Myc antibody. Anti-POLA1 was used to detect POLA1 in precipitates. Two independent co-IP experiments were performed for each mutant to ensure reproducibility.