Human CST abundance determines recovery from diverse forms of DNA damage and replication stress

Abstract

Mammalian CST (CTC1-STN1-TEN1) is a telomere-associated complex that functions in telomere duplex replication and fill-in synthesis of the telomeric C-strand following telomerase action. CST also facilitates genome-wide replication recovery after HU-induced fork stalling by increasing origin firing. CTC1 and STN1 were originally isolated as a DNA polymerase a stimulatory factor. Here we explore how CST abundance affects recovery from drugs that cause different types of DNA damage and replication stress. We show that recovery from HU and aphidicolin induced replication stress is increased by CST over-expression. Elevated CST increases dNTP incorporation and origin firing after HU release and decreases the incidence of anaphase bridges and micronuclei after aphidicolin removal. While the frequency of origin firing after HU release is proportional to CST abundance, the number of cells entering S-phase to initiate replication is unchanged by CST overexpression or STN1 depletion. Instead the CST-related changes in origin firing take place in cells that were already in S-phase at the time of HU addition, indicating that CST modulates firing of late or dormant origins. CST abundance also influences cell viability after treatment with HU, aphidicolin, MMS and camptothecin. Viability is increased by elevated CST and decreased by STN1 depletion, indicating that endogenous CST levels are limiting. However, CST abundance does not affect viability after MMC treatment. Thus, CST facilitates recovery from many, but not all, forms of exogenous DNA damage. Overall our results suggest that CST is needed in stoichiometric amounts to facilitate re-initiation of DNA replication at repaired forks and/or dormant origins.

Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress

“…The CST complex has been shown to be important for reinitiating stalled DNA synthesis at both telomeric and non-telomeric sequences (Gu et al, 2012; Huang et al, 2012; Stewart et al, 2012; Wang et al, 2014). Although it has been proposed that CST promotes origin firing upon fork stalling (Stewart et al, 2012), the molecular mechanism underlying CST-mediated replication re-initiating is largely unclear.…”

“…Originally discovered as a telomere maintenance factor (see below), the human CST complex also promotes efficient replication of difficult-to-replicate sequences in the genome (Kasbek et al, 2013; Stewart et al, 2012). Deficiency in CST components reduces cell viability after exposure to reagents stalling replication forks including HU, aphidicolin (APH), methyl methanesulfonate (MMS) and camptothecin (Wang et al, 2014; Zhou and Chai). Mutations in CTC1 cause Coats Plus disease, a complex disorder characterized by bilateral exudative retinopathy, retinal telangiectasias, growth retardation, intracranial calcifications, bone abnormalities, gastrointestinal vascular ectasias, accompanied by common early-aging pathological features like premature hair graying, anemia, and osteoporosis (Anderson et al, 2012; Armanios and Blackburn, 2012; Keller et al, 2012; Polvi et al, 2012).…”

“…Human CST interacts with the TPP1-POT1 shelterin complex (Chen et al, 2012; Wan et al, 2009). Mammalian CST promotes efficient replication of telomeric DNA, mediates C-strand synthesis at telomere ends during the late S/G2 phase, and inhibits telomerase access to telomeres to prevent excessive telomere lengthening (Chen et al, 2012; Huang et al, 2012; Kasbek et al, 2013; Stewart et al, 2012; Wang et al, 2014). A subset of CTC1 mutations identified in Coats Plus patients induce accelerated telomere shortening and display telomeric DNA replication defects (Anderson et al, 2012; Chen et al, 2013; Gu and Chang, 2013), leading to the conclusion that the pathogenesis of Coats Plus may in part derive from telomere maintenance defects.…”

MTV, an ssDNA Protecting Complex Essential for Transposon-Based Telomere Maintenance in Drosophila

“…In fact the CST complex has been described as a telomeric RPA-like complex based on structural similarities of the two [31; 32]. In addition, CST in mammals is important not only for telomere maintenance but also for genomic replication [33]. Whether the Drosophila MTV complex has a similar role in regulating genome-wide replication remains to be studied.…”

The molecular genetics of the telomere biology disorders

“…This requires the binding of CTC1 to single-stranded DNA and its association with the replication initiator pol primase complex. While these properties suggest that CTC1 might also have a role in general genomic DNA replication, this is restricted to regions experiencing replication stress, where it stimulates the firing of late or dormant origins 105,106 .…”