Guardians of the Genome: How the Single-Stranded DNA-Binding Proteins RPA and CST Facilitate Telomere Replication.

Telomeres act as the protective caps of eukaryotic linear chromosomes; thus, proper telomere maintenance is crucial for genome stability. Successful telomere replication is a cornerstone of telomere length regulation, but this process can be fraught due to the many intrinsic challenges telomeres pose to the replication machinery. In addition to the famous "end replication" problem due to the discontinuous nature of lagging strand synthesis, telomeres require various telomere-specific steps for maintaining the proper 3' overhang length. Bulk telomere replication also encounters its own difficulties as telomeres are prone to various forms of replication roadblocks. These roadblocks can result in an increase in replication stress that can cause replication forks to slow, stall, or become reversed. Ultimately, this leads to excess single-stranded DNA (ssDNA) that needs to be managed and protected for replication to continue and to prevent DNA damage and genome instability. RPA and CST are single-stranded DNA-binding protein complexes that play key roles in performing this task and help stabilize stalled forks for continued replication. The interplay between RPA and CST, their functions at telomeres during replication, and their specialized features for helping overcome replication stress at telomeres are the focus of this review.

RPA engages telomeric G-quadruplexes more effectively than CST.

G-quadruplexes (G4s) are a set of stable secondary structures that form within guanine-rich regions of single-stranded nucleic acids that pose challenges for DNA maintenance. The G-rich DNA sequence at telomeres has a propensity to form G4s of various topologies. The human protein complexes Replication Protein A (RPA) and CTC1-STN1-TEN1 (CST) are implicated in managing G4s at telomeres, leading to DNA unfolding and allowing telomere replication to proceed. Here, we use fluorescence anisotropy equilibrium binding measurements to determine the ability of these proteins to bind various telomeric G4s. We find that the ability of CST to specifically bind G-rich ssDNA is substantially inhibited by the presence of G4s. In contrast, RPA tightly binds telomeric G4s, showing negligible changes in affinity for G4 structure compared to linear ssDNAs. Using a mutagenesis strategy, we found that RPA DNA-binding domains work together for G4 binding, and simultaneous disruption of these domains reduces the affinity of RPA for G4 ssDNA. The relative inability of CST to disrupt G4s, combined with the greater cellular abundance of RPA, suggests that RPA could act as a primary protein complex responsible for resolving G4s at telomeres.

CST does not evict elongating telomerase but prevents initiation by ssDNA binding.

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.