Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1‐STN1‐TEN1) proteins, which form a single‐stranded DNA‐binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent‐strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5’ DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA‐binding ability of CST is required for blocking MRE11‐mediated nascent‐strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent‐strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.
Replication stress causes replication fork stalling, resulting in an accumulation of single-stranded DNA (ssDNA). Replication protein A (RPA) and CTC1-STN1-TEN1 (CST) complex bind ssDNA and are found at stalled forks, where they regulate RAD51 recruitment and foci formation in vivo. Here, we investigate crosstalk between RPA, CST, and RAD51. We show that CST and RPA localize in close proximity in cells. Although CST stably binds to ssDNA with a high affinity at low ionic strength, the interaction becomes more dynamic and enables facilitated dissociation at high ionic strength. CST can coexist with RPA on the same ssDNA and target RAD51 to RPA-coated ssDNA. Notably, whereas RPA-coated ssDNA inhibits RAD51 activity, RAD51 can assemble a functional filament and exhibit strand-exchange activity on CST-coated ssDNA at high ionic strength. Our findings provide mechanistic insights into how CST targets and tethers RAD51 to RPA-coated ssDNA in response to replication stress.
The long non-coding telomeric RNA transcript TERRA, in the form of an RNA–DNA duplex, regulates telomere recombination. In a screen for nucleases that affects telomere recombination, mutations in DNA2, EXO1, MRE11 and SAE2 cause severe delay in type II survivor formation, indicating that type II telomere recombination is mediated through a mechanism similar to repairing double-strand breaks. On the other hand, mutation in RAD27 results in early formation of type II recombination, suggesting that RAD27 acts as a negative regulator in telomere recombination. RAD27 encodes a flap endonuclease that plays a role in DNA metabolism, including replication, repair and recombination. We demonstrate that Rad27 suppresses the accumulation of the TERRA-associated R-loop and selectively cleaves TERRA of R-loop and double-flapped structures in vitro. Moreover, we show that Rad27 negatively regulates single-stranded C-rich telomeric DNA circles (C-circles) in telomerase-deficient cells, revealing a close correlation between R-loop and C-circles during telomere recombination. These results demonstrate that Rad27 participates in telomere recombination by cleaving TERRA in the context of an R-loop or flapped RNA–DNA duplex, providing mechanistic insight into how Rad27 maintains chromosome stability by restricting the accumulation of the R-loop structure within the genome.
Homologous recombination (HR) is an error-free repair pathway to eliminate DNA double-strand breaks and cells with deficiency of HR repair are highly sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Thus, targeting HR-defective cells becomes a new therapeutic approach for cancer patients. HR deficiency can be identified as BRCAness which may be due to mutations or epigenetic silencing in genes involved in HR, such as BRCA1 and BRCA2. Recent studies have developed several approaches to analyze HR status of cancer cells including RAD51 focus formation assay, large-scale mutational signatures analysis, and genome-wide transcriptome profiling. However, these assays provide an indirect estimation of HR status. We wish to directly measure HR rate in cancer cells. We have successfully developed two approaches, plasmid-based and virus-based functional assays, to directly quantify HR activity in cells and serve as a functional biomarker. Our results show that the signal of the functional biomarker specifically depends on the presence of the key enzyme, RAD51 recombinase, in HR. Finally, these functional assays can be utilized in different cancer cell types to determine the HR activity and can be a functional biomarker to evaluate the “BRCAness” status for the treatment of PARP inhibitors. Citation Format: Chih-Ying Lee, Min-Yu Ko, Kai-Hang Lei, Peter Chi. Detection of BRCAness by a functional biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 334.
Homologous recombination (HR) is an error-free repair pathway to eliminate DNA double-strand breaks and crucial for maintaining genome stability. Pathogenic mutations in genes involved in HR, such as BRCA1 and BRCA2, lead to homologous recombination deficiency (HRD) and sensitive cells to poly(ADP-ribose) polymerase inhibitors (PARPi). As such, the selection of HRD cancer patients becomes an important clinical need. In addition to sequencing HR-related genes, recent studies have developed several approaches to detect HRD including genomic scar score, mutational signatures analysis, and RAD51 foci formation assay. However, these assays are not on a real-time basis and provide an indirect estimation of HR status. To overcome these limitations, we have successfully developed a virus-based functional assay to directly quantify HR activity in cells. Our method detects HR status in a real-time and tumor-only manner. By using this activity-based functional assay, we reveal a universal activity threshold for identifying HRD across cancer types. Here, we present a promising method to accurately detect primary ovarian cancer cells with HRD. Clinical samples that be quantified as HRD show a significant response to PARPi. Therefore, our method can serve as a functional biomarker and companion diagnostic for PARPi. Citation Format: Chih-Ying Lee, Kai-Hang Lei, Shih-Han Huang, Min-Yu Ko, Ko-Yu Chang, Po-Han Lin, Yu-Li Chen, Wen-Fang Cheng, Peter Chi. Detection of homologous recombination deficiency across cancer types by a real-time activity-based functional biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 365.
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