Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent‐strand DNA

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. Synopsis The human CTC1‐STN1‐TEN1 (CST) complex binds to ssDNA and has been implicated in protecting genomic stability under replication stress. This study shows that the CST complex localizes to replication forks in response to their stalling, and protects them from degradation by MRE11 nuclease. Human CST proteins localizes at stalled replication forks. CST deficiency increases MRE11 association with stalled forks and MRE11‐mediated degradation of nascent‐strand DNA at reversed forks, leading to genome instability. The CST complex binds to ds/ssDNA substrates and directly blocks MRE11 degradation of DNA in a sequence‐independent manner. The DNA binding activity of CST is a prerequisite for blocking MRE11 degradation at reversed forks. CST inactivation exacerbates genome instability in BRCA2‐deficient cells. Graphical Abstract CTC1‐STN1‐TEN1, originally identified as telomeric single‐strand binding proteins, join the ranks of fork protecting factors, by virtue of ss/dsDNA junction binding in response to replication stalling.