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Abstract 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. 

Replication protein A (RPA) is a ubiquitous ssDNAbinding protein that functions in many DNA processing pathways to maintain genome integrity. Recent studies suggest that RPA forms a highly dynamic complex with ssDNA that can engage with DNA in many modes that are orchestrated by the differential engagement of the four DNA-binding domains (DBDs) in RPA. To understand how these modes influence RPA interaction with biologically relevant ligands, we performed a comprehensive and systematic evaluation of RPA’s binding to a diverse set of ssDNA ligands that varied in sequence, length, and structure. These equilibrium binding data show that WT RPA binds structured ssDNA ligands differently from its engagement with minimal ssDNAs. Next, we investigated each DBD’s contributions to RPA’s binding modes through mutation of conserved, functionally important aromatic residues. Mutations in DBDA and -B have a much larger effect on binding when ssDNA is embedded into DNA secondary structures compared to their association with unstructured minimal ssDNA. As our data support a complex interplay of binding modes, it is critical to define the trimer core DBDs’ role in binding these biologically relevant ligands. We found that DBD-C is important for engaging DNA with diverse binding modes, including, unexpectedly, at short ssDNAs. Thus, RPA uses its constituent DBDs to bind biologically diverse ligands in unanticipated ways. These findings lead to a better understanding of how RPA carries out its functions at diverse locations of the genome and suggest a mechanism through which dynamic recognition can impact differential downstream outcomes. 

Estrogen receptor alpha (ERα) is a ligand-responsive transcription factor critical for sex determination and development. Recent reports challenge the canonical view of ERα function by suggesting an activity beyond binding dsDNA at estrogen-responsive promotor elements: association with RNAs in vivo. Whether these interactions are direct or indirect remains unknown, which limits the ability to understand the extent, specificity, and biological role of ERα-RNA binding. Here we demonstrate that an extended DNA-binding domain of ERα directly binds a wide range of RNAs in vitro with structural specificity. ERα binds RNAs that adopt a range of hairpin-derived structures independent of sequence, while interacting poorly with single- and double-stranded RNA. RNA affinities are only 4-fold weaker than consensus dsDNA and significantly tighter than nonconsensus dsDNA sequences. Moreover, RNA binding is competitive with DNA binding. Together, these data show that ERα utilizes an extended DNA-binding domain to achieve a high-affinity/low-specificity mode for interacting with RNA. 

Cyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif ( AAUAAUAA ) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1. 

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo–electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome’s organization of double-stranded DNA.