STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

Abstract

Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress.

Human CST Prefers G-Rich but Not Necessarily Telomeric Sequences

“…22,34 Also analogous to RPA, CST appears to have two binding modes, a short, specific one and a longer, nonspecific mode. 44,45 We focused on the specific binding mode, as the longer, nonspecific mode is likely achieved through plasticity at the protein– ssDNA interface due to the inherent flexibility of the ssDNA ligand. The precise minimal length for specific binding is not known.…”

Shared Subunits of Tetrahymena Telomerase Holoenzyme and Replication Protein A Have Different Functions in Different Cellular Complexes

“…Vertebrate CST contributes to DNA replication at sites throughout the genome and, with distinct structural requirements, to telomere-specific processes, such as the post-replication cytidine-rich strand (C-strand) fill-in by polymerase ␣-primase (28 -30). Vertebrate CST also has been proposed to inhibit telomerase access to chromosome ends, although this role is not uniformly evident across different studies (30,31). Budding yeast CST function is telomere-specific; it stimulates C-strand fill-in and contributes to chromosome end-capping, and, when disassembled in S-phase, its Cdc13 subunit recruits telomerase holoenzyme (18).…”

Sequence, structure and evolutionary analysis of cold shock domain proteins, a member of OB fold family

“…Studies show that different OB folds can bind and establish protein-DNA, protein-RNA and proteinprotein interactions (Arcus, 2002;Agrawal & Kishan, 2003;Theobald et al, 2003). Among these, the interaction of OB fold with ssDNA is most extensively studied and characterized (Theobald et al, 2003;Dickey & Wuttke, 2014;Bhattacharjee et al, 2016;Wu et al, 2016;Gu et al, 2017;Shastrula et al, 2017).…”