Abstract: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 ss… Show more
“…CTC1 and STN1 mutations have been implicated in the telomere-related Coats Plus syndrome and patients with CTC1 mutations exhibit telomere dysfunction that is consistent with telomeric DNA replication errors ( Anderson et al, 2012 ; Chen et al, 2013 ; Simon A. J. et al, 2016 ). Importantly and in contrast to budding yeast, mammalian CST and the ST complex in S. pombe also appear to have extratelomeric functions in DNA replication and fork restart under conditions of replication stress that are outside the scope of this review ( Price et al, 2010 ; Stewart et al, 2012 ; Wang et al, 2014 , 2019 ; Lyu et al, 2021 ).…”
Section: Telomeric Dna Replication By the Conventional Replication Mamentioning
Eukaryotic genomes are highly complex and divided into linear chromosomes that require end protection from unwarranted fusions, recombination, and degradation in order to maintain genomic stability. This is accomplished through the conserved specialized nucleoprotein structure of telomeres. Due to the repetitive nature of telomeric DNA, and the unusual terminal structure, namely a protruding single stranded 3′ DNA end, completing telomeric DNA replication in a timely and efficient manner is a challenge. For example, the end replication problem causes a progressive shortening of telomeric DNA at each round of DNA replication, thus telomeres eventually lose their protective capacity. This phenomenon is counteracted by the recruitment and the activation at telomeres of the specialized reverse transcriptase telomerase. Despite the importance of telomerase in providing a mechanism for complete replication of telomeric ends, the majority of telomere replication is in fact carried out by the conventional DNA replication machinery. There is significant evidence demonstrating that progression of replication forks is hampered at chromosomal ends due to telomeric sequences prone to form secondary structures, tightly DNA-bound proteins, and the heterochromatic nature of telomeres. The telomeric loop (t-loop) formed by invasion of the 3′-end into telomeric duplex sequences may also impede the passage of replication fork. Replication fork stalling can lead to fork collapse and DNA breaks, a major cause of genomic instability triggered notably by unwanted repair events. Moreover, at chromosomal ends, unreplicated DNA distal to a stalled fork cannot be rescued by a fork coming from the opposite direction. This highlights the importance of the multiple mechanisms involved in overcoming fork progression obstacles at telomeres. Consequently, numerous factors participate in efficient telomeric DNA duplication by preventing replication fork stalling or promoting the restart of a stalled replication fork at telomeres. In this review, we will discuss difficulties associated with the passage of the replication fork through telomeres in both fission and budding yeasts as well as mammals, highlighting conserved mechanisms implicated in maintaining telomere integrity during replication, thus preserving a stable genome.
“…CTC1 and STN1 mutations have been implicated in the telomere-related Coats Plus syndrome and patients with CTC1 mutations exhibit telomere dysfunction that is consistent with telomeric DNA replication errors ( Anderson et al, 2012 ; Chen et al, 2013 ; Simon A. J. et al, 2016 ). Importantly and in contrast to budding yeast, mammalian CST and the ST complex in S. pombe also appear to have extratelomeric functions in DNA replication and fork restart under conditions of replication stress that are outside the scope of this review ( Price et al, 2010 ; Stewart et al, 2012 ; Wang et al, 2014 , 2019 ; Lyu et al, 2021 ).…”
Section: Telomeric Dna Replication By the Conventional Replication Mamentioning
Eukaryotic genomes are highly complex and divided into linear chromosomes that require end protection from unwarranted fusions, recombination, and degradation in order to maintain genomic stability. This is accomplished through the conserved specialized nucleoprotein structure of telomeres. Due to the repetitive nature of telomeric DNA, and the unusual terminal structure, namely a protruding single stranded 3′ DNA end, completing telomeric DNA replication in a timely and efficient manner is a challenge. For example, the end replication problem causes a progressive shortening of telomeric DNA at each round of DNA replication, thus telomeres eventually lose their protective capacity. This phenomenon is counteracted by the recruitment and the activation at telomeres of the specialized reverse transcriptase telomerase. Despite the importance of telomerase in providing a mechanism for complete replication of telomeric ends, the majority of telomere replication is in fact carried out by the conventional DNA replication machinery. There is significant evidence demonstrating that progression of replication forks is hampered at chromosomal ends due to telomeric sequences prone to form secondary structures, tightly DNA-bound proteins, and the heterochromatic nature of telomeres. The telomeric loop (t-loop) formed by invasion of the 3′-end into telomeric duplex sequences may also impede the passage of replication fork. Replication fork stalling can lead to fork collapse and DNA breaks, a major cause of genomic instability triggered notably by unwanted repair events. Moreover, at chromosomal ends, unreplicated DNA distal to a stalled fork cannot be rescued by a fork coming from the opposite direction. This highlights the importance of the multiple mechanisms involved in overcoming fork progression obstacles at telomeres. Consequently, numerous factors participate in efficient telomeric DNA duplication by preventing replication fork stalling or promoting the restart of a stalled replication fork at telomeres. In this review, we will discuss difficulties associated with the passage of the replication fork through telomeres in both fission and budding yeasts as well as mammals, highlighting conserved mechanisms implicated in maintaining telomere integrity during replication, thus preserving a stable genome.
“…Overall then, the CST complex plays an important role in protecting and restarting stalled replication forks. Importantly, depletion of CST resulted in genome instabilities similar to those displayed by BRCA2-depleted cells 14 , 31 . Mechanistically, we have provided evidence that CST localizes at stalled replication forks upon hydroxyurea treatment and that CST depletion causes MRE11-mediated degradation of nascent strand DNA at reversed forks, with purified CST complex blocking degradation of DNA by MRE11 in vitro 31 .…”
Section: Introductionmentioning
confidence: 88%
“…It is worth noting that RAD51 can catalyze the preformed RPA-ssDNA substrate for strand exchange at higher concentrations 23 , 28 , 29 . Our recent in situ protein interactions at nascent and stalled replication forks (SIRF) analysis 30 demonstrates that the CST complex facilitates recruitment of RAD51 to stalled forks upon hydroxyurea treatment 31 , 32 . Fig.…”
Section: Introductionmentioning
confidence: 99%
“…Previous work by us and others has documented that the CST complex participates in reinitiating stalled DNA synthesis at both telomeric and non-telomeric sequences 32 , 36 , 37 , 41 , 45 . DNA fiber analysis has further evidenced that STN1 or CTC1 depletion enhanced fork degradation and that RNAi-resistant wild-type STN1 or CTC1, respectively, rescued that phenotype 31 . Overall then, the CST complex plays an important role in protecting and restarting stalled replication forks.…”
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.
“…CST facilitates RAD51 recruitment to forks stalled at GC-rich repetitive sequences and is important for the stability of these sequences [18]. In addition, CST binding to reversed forks can directly block MRE11 nuclease degradation of nascent strand DNA, thereby protecting fork stability [19].…”
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
