An official website of the United States government
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.
more »
« less
Shelterin reduces the accessibility of telomeric overhangs
Abstract Telomeres terminate with a 50–300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.
more »
« less
- Award ID(s):
- 1954449
- PAR ID:
- 10385239
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 22
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 12885-12895
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract 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 might bring the distance between PNA amide groups closer to 7 Å, which is favourable for hydrogen bonding to the B‐form dsDNA phosphate oxygens. Extension of the 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 PNAs might have an inherent preference for A‐form‐like conformations when binding double‐stranded nucleic acids. It appears that the original six‐atom‐long PNA backbone is an almost perfect fit for binding to A‐form nucleic acids.more » « less
-
Abstract Single-stranded DNA binding proteins (SSBs) avidly bind ssDNA and yet enzymes that need to act during DNA replication and repair are not generally impeded by SSB, and are often stimulated by SSB. Here, the effects of Escherichia coli SSB on the activities of the DNA polymerase processivity clamp loader were investigated. SSB enhances binding of the clamp loader to DNA by increasing the lifetime on DNA. Clamp loading was measured on DNA substrates that differed in length of ssDNA overhangs to permit SSB binding in different binding modes. Even though SSB binds DNA adjacent to single-stranded/double-stranded DNA junctions where clamps are loaded, the rate of clamp loading on DNA was not affected by SSB on any of the DNA substrates. Direct measurements of the relative timing of DNA-SSB remodeling and enzyme–DNA binding showed that the clamp loader rapidly remodels SSB on DNA such that SSB has little effect on DNA binding rates. However, when SSB was mutated to reduce protein–protein interactions with the clamp loader, clamp loading was inhibited by impeding binding of the clamp loader to DNA. Thus, protein–protein interactions between the clamp loader and SSB facilitate rapid DNA-SSB remodeling to allow rapid clamp loader-DNA binding and clamp loading.more » « less
-
Zappulla, David C (Ed.)TRF2 is an essential and conserved double-strand telomere binding protein that stabilizes chromosome ends by suppressing DNA damage response and aberrant DNA repair. Herein we investigated the mechanisms and functions of the Trf2 ortholog in the basidiomycete fungusUstilago maydis, which manifests strong resemblances to metazoans with regards to the telomere and DNA repair machinery. We showed thatUmTrf2 binds to Blmin vitroand inhibits Blm-mediated unwinding of telomeric DNA substrates. Consistent with a similar inhibitory activityin vivo, over-expression of Trf2 induces telomere shortening, just like deletion ofblm, which is required for efficient telomere replication. While the loss of Trf2 engenders growth arrest and multiple telomere aberrations, these defects are fully suppressed by the concurrent deletion ofblmormre11(but not other DNA repair factors). Over-expression of Blm alone triggers aberrant telomere recombination and the accumulation of aberrant telomere structures, which are blocked by concurrent Trf2 over-expression. Together, these findings highlight the suppression of Blm as a key protective mechanism of Trf2. Notably,U.maydisharbors another double-strand telomere-binding protein (Tay1), which promotes Blm activity to ensure efficient replication. We found that deletion oftay1partially suppresses the telomere aberration of Trf2-depleted cells. Our results thus point to opposing regulation of Blm helicase by telomere proteins as a strategy for optimizing both telomere maintenance and protection. We also show that aberrant transcription of both telomere G- and C-strand is a recurrent phenotype of telomere mutants, underscoring another potential similarity between double strand breaks and de-protected telomeres.more » « less
-
Telomeres cap chromosome ends with specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. Fission yeast telomeres can be formed by cleaving a “proto-telomere” bearing 48 bp of telomere repeats to form a new stable chromosomal end that prevents the rapid degradation seen at similar DNA double-strand breaks (DSBs). This end-protection was investigated in viable mutants lacking telomere-associated proteins. Telomerase, the shelterin components Taz1, Rap1, or Poz1 or the telomere-associated protein Rif1 were not required to form a stable chromosome end after cleavage of the proto-telomere. However, cells lacking the fission yeast shelterin component Ccq1 converted the cleaved telomere repeat-capped end to a rapidly degraded DSB. Degradation was greatly reduced by eliminating the nuclease activity of Mre11, a component of the Mre11-Rad50-Nbs1/Xrs2 complex that processes DSBs. These results demonstrate a novel function for Ccq1 to effect end-protection by restraining Mre11-dependent degradation.more » « less
