Human CST complex protects replication fork stability by directly blocking MRE11 degradation of nascent strand DNA

Abstract

Degradation and collapse of stalled replication forks are main sources of genome instability, yet the molecular mechanism for protecting forks from degradation/collapse is not well understood. Here, we report that human CST (CTC1-STN1-TEN1), a single-stranded DNA binding protein complex, localizes at stalled forks and protects forks from MRE11 nuclease degradation upon replication perturbation. CST deficiency causes nascent strand degradation, ssDNA accumulation after fork stalling, and delay in replication recovery, leading to cellular sensitivity to fork stalling agents. Purified CST binds to 5' overhangs and directly blocks MRE11 degradation in vitro, and the DNA binding ability of CST is required for blocking MRE11-mediated nascent strand degradation. Finally, we uncover that CST and BRCA2 form non-overlapping foci upon fork stalling, and CST inactivation is synthetic with BRCA2 deficiency in inducing genome instability. Collectively, our findings identify CST as an important fork protector to preserve genome integrity under replication perturbation.

Roles of OB-Fold Proteins in Replication Stress

“…The low abundance of CST may partially explain why iPOND has not been successful in detecting CST at stalled forks. Using the SIRF ( in situ protein interactions at nascent and stalled replication forks) assay, we are able to detect CST at stalled forks ( Lyu et al, 2019b ), thus providing direct evidence that CST also localizes at stalled forks. Many questions remain to be answered in order to fully understand the genome maintenance mechanisms in response to fork stalling.…”

“…Suppression of each CST subunit impairs HU-induced RAD51 foci formation as well as RAD51 binding to GC-rich repetitive sites, suggesting that CST may facilitate the recruitment of RAD51 to stalled sites after HU-induced RS ( Chastain et al, 2016 ; Figure 4C ). CST is also recently shown to be localized at stalled replication fork and stabilize the fork by blocking MRE11-mediated nascent strand degradation ( Lyu et al, 2019b ; Figure 4C ). These findings provide a mechanistic link between CST and other key players in fork stabilization and fork restart, at least at G-rich sequences.…”

“…Unlike RPA and BRCA2, CST is a relatively new member in genome maintenance. Knockdown of CTC1 or STN1 elevates the level of multi-telomeric signals and telomere instabilities ( Surovtseva et al, 2009 ; Dai et al, 2010 ) and increases the formation of anaphase bridges, micronuclei, chromosome breakage, and chromosome pulverization ( Stewart et al, 2012 ; Chastain et al, 2016 ; Lyu et al, 2019b ). Disease-causing CTC1 mutations induce spontaneous chromosome instabilities that are further increased by RS ( Wang and Chai, 2018 ).…”

“…If they are in parallel pathways, do they protect forks stalled at different regions in the genome? While it is tempting to speculate that CST may be a RAD51 mediator by displacing RPA from ssDNA in a manner similar to BRCA2-DSS1, it has been reported that the DNA-binding ability of BRCA2 is dispensable for FP ( Schlacher et al, 2011 ) while CST binding to DNA is required for FP ( Lyu et al, 2019b ), suggesting that there may be a fundamental difference underlying FP mechanisms by BRCA2 and CST. In addition, CST differs from BRCA2 in that it mediates POLα fill-in synthesis at telomere ends ( Dai et al, 2010 ; Huang et al, 2012 ), and it has been proposed that CST/POLα-dependent fill-in synthesis may counteract end resection at DSB ends ( Mirman et al, 2018 ).…”

“…RPA participates in RS response via its binding to ssDNA ( Wold, 1997 ; Zou et al, 2006 ; Glanzer et al, 2014 ; Belanger et al, 2018 ). Structure of the OB-A domain of RPA70 was solved early and has been used for characterizing other OB-fold proteins ( Bochkarev et al, 1997 ), including BRCA2 and CST that have been implicated in RS response ( Yang et al, 2002 ; Bochkarev and Bochkareva, 2004 ; Sun et al, 2009 ; Wang and Chai, 2018 ; Lyu et al, 2019b ). Through binding to ssDNA and their protein binding partners at stalled forks via OB-fold domains, these proteins influence the remodeling of stalled forks, modulate the activities of other important proteins at forks, and/or act as signal responders to fork stalling.…”