Dynamic DNA binding, junction recognition and G4 melting activity underlie the telomeric and genome-wide roles of human CST

Abstract

Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that helps resolve replication problems both at telomeres and genome-wide. CST resembles Replication Protein A (RPA) in that the two complexes harbor comparable arrays of OB-folds and have structurally similar small subunits. However, the overall architecture and functions of CST and RPA are distinct. Currently, the mechanism underlying CST action at diverse replication issues remains unclear. To clarify CST mechanism, we examined the capacity of CST to bind and resolve DNA structures found at sites of CST activity. We show that CST binds preferentially to ss-dsDNA junctions, an activity that can explain the incremental nature of telomeric C-strand synthesis following telomerase action. We also show that CST unfolds G-quadruplex structures, thus providing a mechanism for CST to facilitate replication through telomeres and other GC-rich regions. Finally, smFRET analysis indicates that CST binding to ssDNA is dynamic with CST complexes undergoing concentration-dependent self-displacement. These findings support an RPA-based model where dissociation and re-association of individual OB-folds allow CST to mediate loading and unloading of partner proteins to facilitate various aspects of telomere replication and genome-wide resolution of replication stress.

CTC1 mutations in a Brazilian family with progeroid features and recurrent bone fractures

“…This is similar to what has been reported in CTC1 deficient mouse embryonic fibroblasts (Gu et al, 2012). Co-immunostaining of 53BP1 and TRF1 showed that the correlations of the sub-nuclear localizations of these proteins were not different among LCLs derived from the patient, heterozygous parents or controls, supporting the notion that genomic instability caused by aberrant CTC1 function is not limited to the telomeres, but extends to additional regions of the genome (Bhattacharjee et al, 2017;Y. Wang & Chai, 2018).…”

CTC1‐STN1 coordinates G‐ and C‐strand synthesis to regulate telomere length

“…However, continuous cellular replication in tissues with limiting DNA Pol‐α levels results in C‐strand maintenance defects, manifested as stalled replication forks unable to bypass G‐rich secondary structures including G‐quadruplexes (G4), resulting in the formation of single‐stranded gaps that when degraded give rise to STL. Both POT1 and CST efficiently disrupt G‐quadruplex formation in vitro (Bhattacharjee, Wang, Diao & Price, 2017; Wang, Nora, Ghodke & Opresko, 2011), and our data suggest that introduction of WT CTC1 into CTC1 L1142H mutants completely suppressed STL formation (Figure 3g). We postulate that CST/POT1 plays an important role in preventing the formation of G4 on ss telomeric G‐rich DNA to maintain genome stability.…”

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

“…MRE11 possesses the 3' to 5' exonuclease activity and can extensively degrade ss-ds DNA junction structures with 5' overhangs formed by regressed forks (Kolinjivadi et al, 2017). It has been reported that CST prefers binding to ss-ds DNA junctions in vitro (Bhattacharjee et al, 2017). Hence, we hypothesized that CST might bind to 5' overhangs of the regressed arms to protect nascent strand DNA from MRE11 degradation.…”

“…It is capable of binding to an 18-nt G-rich ssDNA with high affinity, but the sequence specificity is lost if the oligonucleotide becomes longer (Hom & Wuttke, 2017;Miyake et al, 2009). Interestingly, although CST is unable to bind to dsDNA, ss-ds DNA junctions stabilize CST binding and decrease minimal ssDNA length requirement for CST binding to 10 nt (Bhattacharjee et al, 2017), indicating that CST may bind to special DNA structures with ss-ds junctions in vivo. A set of missense mutations of CTC1 and STN1 genes have been identified in patients with the Coats plus disease, a rare autosomal recessive disorder characterized by bilateral exudative retinopathy, retinal telangiectasias, growth retardation, intracranial calcifications, bone abnormalities, gastrointestinal vascular ectasias, accompanied by common early-aging pathological features (Anderson et al, 2012;Keller et al, 2012;Simon et al, 2016).…”