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1. Slow G-Quadruplex Conformation Rearrangement and Accessibility Change Induced by Potassium in Human Telomeric Single-Stranded DNA

   https://doi.org/10.1021/acs.jpcb.4c00719  

   Lacen, Arianna N; Symasek, Andrew; Gunter, Alan; Lee, Hui-Ting (June 2024, The Journal of Physical Chemistry B)

   The guanine-rich telomeric repeats can form G-quadruplexes (G4s) that alter the accessibility of the single-stranded telomeric overhang. In this study, we investigated the effects of Na+ and K+ on G4 folding and accessibility through cation introduction and exchange. We combined differential scanning calorimetry (DSC), circular dichroism (CD), and single molecule Förster resonance energy transfer (smFRET) to monitor the stability, conformational dynamics, and complementary strand binding accessibility of G4 formed by single-stranded telomeric DNA. Our data showed that G4 formed through heating and slow cooling in K+ solution exhibited fewer conformational dynamics than G4 formed in Na+ solution, which is consistent with the higher thermal stability of G4 in K+. Monitoring cation exchange with real time smFRET at room temperature shows that Na+ and K+ can replace each other in G4. When encountering high K+ at room or body temperature, G4 undergoes a slow conformational rearrangement process which is mostly complete by 2 h. The slow conformational rearrangement ends with a stable G4 that is unable to be unfolded by a complementary strand. This study provides new insights into the accessibility of G4 forming sequences at different time points after introduction to a high K+ environment in cells, which may affect how the nascent telomeric overhang interacts with proteins and telomerase. 

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2. Ustilago maydis telomere protein Pot1 harbors an extra N-terminal OB fold and regulates homology-directed DNA repair factors in a dichotomous and context-dependent manner

   https://doi.org/10.1371/journal.pgen.1010182  

   Zahid, Syed; Aloe, Sarah; Sutherland, Jeanette H.; Holloman, William K.; Lue, Neal F. (May 2022, PLOS Genetics)

   Zhou, Jin-Qiu (Ed.)

   The telomere G-strand binding protein Pot1 plays multifaceted roles in telomere maintenance and protection. We examined the structure and activities of Pot1 in Ustilago maydis , a fungal model that recapitulates key features of mammalian telomere regulation. Compared to the well-characterized primate and fission yeast Pot1 orthologs, Um Pot1 harbors an extra N-terminal OB-fold domain (OB-N), which was recently shown to be present in most metazoans. Um Pot1 binds directly to Rad51 and regulates the latter’s strand exchange activity. Deleting the OB-N domain, which is implicated in Rad51-binding, caused telomere shortening, suggesting that Pot1-Rad51 interaction facilitates telomere maintenance. Depleting Pot1 through transcriptional repression triggered growth arrest as well as rampant recombination, leading to multiple telomere aberrations. In addition, telomere repeat RNAs transcribed from both the G- and C-strand were dramatically up-regulated, and this was accompanied by elevated levels of telomere RNA-DNA hybrids. Telomere abnormalities of pot1 -deficient cells were suppressed, and cell viability was restored by the deletion of genes encoding Rad51 or Brh2 (the BRCA2 ortholog), indicating that homology-directed repair (HDR) proteins are key mediators of telomere aberrations and cellular toxicity. Together, these observations underscore the complex physical and functional interactions between Pot1 and DNA repair factors, leading to context-dependent and dichotomous effects of HDR proteins on telomere maintenance and protection. 

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3. The Maize Single myb histone 1 Gene, Smh1 , Belongs to a Novel Gene Family and Encodes a Protein That Binds Telomere DNA Repeats in Vitro

   https://doi.org/10.1104/pp.103.026856  

   Marian, Calin O; Bordoli, Stefano J; Goltz, Marion; Santarella, Rachel A; Jackson, Leisa P; Danilevskaya, Olga; Beckstette, Michael; Meeley, Robert; Bass, Hank W (November 2003, Plant Physiology)

   Abstract We screened maize (Zea mays) cDNAs for sequences similar to the single myb-like DNA-binding domain of known telomeric complex proteins. We identified, cloned, and sequenced five full-length cDNAs representing a novel gene family, and we describe the analysis of one of them, the gene Single myb histone 1 (Smh1). The Smh1 gene encodes a small, basic protein with a unique triple motif structure of (a) an N-terminal SANT/myb-like domain of the homeodomain-like superfamily of 3-helical-bundle-fold proteins, (b) a central region with homology to the conserved H1 globular domain found in the linker histones H1/H5, and (c) a coiled-coil domain near the C terminus. The Smh-type genes are plant specific and include a gene family in Arabidopsis and the PcMYB1 gene of parsley (Petroselinum crispum) but are distinct from those (AtTRP1, AtTBP1, and OsRTBP1) recently shown to encode in vitro telomere-repeat DNA-binding activity. The Smh1 gene is expressed in leaf tissue and maps to chromosome 8 (bin 8.05), with a duplicate locus on chromosome 3 (bin 3.09). A recombinant full-length SMH1, rSMH1, was found by band-shift assays to bind double-stranded oligonucleotide probes with at least two internal tandem copies of the maize telomere repeat, TTTAGGG. Point mutations in the telomere repeat residues reduced or abolished the binding, whereas rSMH1 bound nonspecifically to single-stranded DNA probes. The two DNA-binding motifs in SMH proteins may provide a link between sequence recognition and chromatin dynamics and may function at telomeres or other sites in the nucleus. 

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4. The structure of human CST reveals a decameric assembly bound to telomeric DNA

   https://doi.org/10.1126/science.aaz9649  

   Lim, Ci Ji; Barbour, Alexandra T.; Zaug, Arthur J.; Goodrich, Karen J.; McKay, Allison E.; Wuttke, Deborah S.; Cech, Thomas R. (June 2020, Science)

   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. 

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5. Triplex‐Forming Peptide Nucleic Acids with Extended Backbones

   https://doi.org/10.1002/cbic.202000432  

   Kumar, Vipin; Brodyagin, Nikita; Rozners, Eriks (July 2020, ChemBioChem)

   Peptide nucleic acid (PNA) forms a triple helix with double‐stranded RNA (dsRNA) stabilized by a hydrogen‐bonding zipper formed by PNA’s backbone amides (N‐H) interacting with RNA phosphate oxygens. This hydrogen‐bonding pattern is enabled by the matching ~5.7 Å spacing (typical for A‐form dsRNA) between PNA’s backbone amides and RNA phosphate oxygens. We hypothesized that extending the PNA’s backbone by one ‐CH2‐ group may bring the distance between PNA amide N‐H closer to 7 Å, favourable for hydrogen‐bonding to the B‐form dsDNA phosphate oxygens. Extension of PNA backbone was expected to selectively stabilize PNA‐DNA triplexes compared to PNA‐RNA. To test this hypothesis, we synthesized triplex‐forming PNAs that had the pseudopeptide backbones extended by an additional ‐CH2‐ group in three different positions. Isothermal titration calorimetry measurements of the binding affinity of these extended PNA analogues for the matched dsDNA and dsRNA showed that, contrary to our structural reasoning, extending the PNA backbone at any position had a strong negative effect on triplex stability. Our results suggest that PNA may have an inherent preference for A‐form‐like conformations when binding double‐stranded nucleic acids. It appears that the original six atoms long PNA backbone is an almost perfect fit for binding to A‐form nucleic acids. 

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