Human CST Prefers G-Rich but Not Necessarily Telomeric Sequences

Abstract

The human CST (CTC1–STN1–TEN1) heterotrimeric complex plays roles in both telomere maintenance and DNA replication through its ability to interact with single-stranded DNA (ssDNA) of a variety of sequences. The precise sequence specificity required to execute these functions is unknown. Telomere-binding proteins have been shown to specifically recognize key telomeric sequence motifs within ssDNA while accommodating nonspecifically recognized sequences through conformationally plastic interfaces. To better understand the role CST plays in these processes, we have produced a highly purified heterotrimer and elucidated the sequence requirements for CST recognition of ssDNA in vitro. CST discriminates against random sequence and binds a minimal ssDNA comprised of three repeats of telomeric sequence. Replacement of individual nucleotides with their complement reveals that guanines are specifically recognized in a largely additive fashion and that specificity is distributed uniformly throughout the ligand. Unexpectedly, adenosines are also well tolerated at these sites, but cytosines are disfavored. Furthermore, sequences unrelated to the telomere repeat, yet still G-rich, bind CST well. Thus, CST is not inherently telomere-specific, but rather is a G-rich sequence binder. This biochemical activity is reminiscent of the yeast t-RPA and Tetrahymena thermophila CST complexes and is consistent with roles at G-rich sites throughout the genome.

Spontaneous replication fork collapse regulates telomere length homeostasis in wild type cells

“…Specificity for chromosome ends is mediated by a high affinity DNA binding domain in Cdc13, which exhibits exceptional sequence preference for single-stranded G-rich telomeric sequences in budding yeast (19) and closely related species (20), leading us to refer to it as a t-RPA complex. This contrasts with the genome-wide role performed by this conserved RPA-like complex in multiple other species (21)(22)(23)(24), consistent with the lack of strong specificity for telomeric DNA (25).…”

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

“…6). Given that CST prefers binding to DNA with specific sequence (G-rich repetitive) and structural features (ss-ds junctions) (Bhattacharjee et al, 2017;Chastain et al, 2016;Hom & Wuttke, 2017) while BRCA2 binds to G-rich oligos with low affinity (Yang et al, 2002), it is possible that CST may be a surrogate of BRCA2 that is particularly important for maintaining the stability of G-rich repetitive sequences and/or sequences prone to form special structures -an interesting hypothesis to be further tested. Alternatively, it remains a possibility that CST and BRCA2 may occupy the same stalled site at different time following fork stalling.…”

“…It has been shown that CST prefers binding to DNA with G-rich repetitive sequences and structural features including ss-ds junctions (Bhattacharjee et al, 2017;Chastain et al, 2016;Hom & Wuttke, 2017). We postulated that CST might localize at forks stalled at non-BRCA2 binding regions and be particularly important for protecting the stabilities of these sequences.…”

“…CST prefers binding to G-rich sequences in vitro in a manner dependent on the sequence length (Chen et al, 2012;Miyake et al, 2009). 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.…”

Mammalian CST averts replication failure by preventing G-quadruplex accumulation

“…Although CST and RPA share many similarities in terms of structure and DNA binding activity, they are architecturally different and they play quite distinct roles during DNA replication and repair [26]. Moreover, CST binds preferentially to G-rich ssDNA and, unlike ssDNA-bound RPA, it does not trigger ATR activation [19,25,27].…”