Novel Centromeric and Subtelomeric Repetitive DNA Sequences for Karyotyping the Bambara Groundnut (Vigna subterranea L. Verdc.)

Bambara groundnut (<i>Vigna subterranea</i> L. Verdc.) is an un­derutilized minor legume crop with climate resilience and great potential use in world agriculture. This study aimed to cytogenetically characterize the genome and chromosome properties of Bambara groundnut. We cloned, sequenced, and mapped a 50-bp centromere-specific tandem repeat on all chromosomes. In addition, a 400-bp subtelomeric repeat was discovered and mapped on a single pair of chromosomes. A Bambara groundnut karyotype was constructed using these novel repeats along with ribosomal RNA genes (45S and 5S) and telomeric DNA sequences. This study provides the first analysis of the genome and chromosome properties of Bambara groundnut. We discuss our findings in relation to genetic improvement of Bambara groundnut and centromere evolution in legume species.

Reconstitution of a telomeric replicon organized by CST

Telomeres, the natural ends of linear chromosomes, are comprised of repeat-sequence DNA and associated proteins1. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells2. Replication begins with telomerase3 extending the single-stranded DNA (ssDNA) of the telomeric G-strand [(TTAGGG)n]; the synthesis of the complementary C-strand [(CCCTAA)n] is much less well characterized. The CST (CTC1-STN1-TEN1) protein complex, a DNA Polymerase α-primase accessory factor4,5, is known to be required for telomere replication in vivo6,7,8,9, and the molecular analysis presented here reveals key features of its mechanism. We find that CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the suboptimality of this template likely contributes to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer, and CST-Polα-primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3’-overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension10,11 and recruits Polα-primase to telomeric ssDNA4,12,13, but it also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.

Structure of telomerase-bound CST with Polymerase α-Primase

Telomeres are the physical ends of linear chromosomes, composed of short repeating sequences (e.g. TTGGGG in Tetrahymena for the G-strand) of double-stranded DNA with a single-strand 3'-overhang of the G-strand and a group of proteins called shelterin. Among these, TPP1 and POT1 associate with the 3'-overhang, with POT1 binding the G-strand and TPP1 recruiting telomerase via interaction with telomerase reverse transcriptase (TERT). The ends of the telomeric DNA are replicated and maintained by telomerase, for the G-strand, and subsequently DNA Polymerase α-Primase (PolαPrim), for the C-strand. PolαPrim is stimulated by CTC1-STN1-TEN1 (CST), but the structural basis of both PolαPrim and CST recruitment to telomere ends remains unknown. Here we report cryo-EM structures of Tetrahymena CST in the context of telomerase holoenzyme, both in the absence and presence of PolαPrim, as well as of PolαPrim alone. Ctc1 binds telomerase subunit p50, a TPP1 ortholog, on a flexible Ctc1 binding motif unveiled jointly by cryo-EM and NMR spectroscopy. PolαPrim subunits are arranged in a catalytically competent conformation, in contrast to previously reported autoinhibited conformation. Polymerase POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. Together, we obtained a snapshot of four key players required for telomeric DNA synthesis in a single complex-telomerase core RNP, p50/TPP1, CST and PolαPrim-that provides unprecedented insights into CST and PolαPrim recruitment and handoff between G-strand and C-strand synthesis.

Structured Waters Mediate Small Molecule Binding to G-Quadruplex Nucleic Acids

The role of G-quadruplexes in human cancers is increasingly well-defined. Accordingly, G-quadruplexes can be suitable drug targets and many small molecules have been identified to date as G-quadruplex binders, some using computer-based design methods and co-crystal structures. The role of bound water molecules in the crystal structures of G-quadruplex-small molecule complexes has been analyzed in this study, focusing on the water arrangements in several G-quadruplex ligand complexes. One is the complex between the tetrasubstituted naphthalene diimide compound MM41 and a human intramolecular telomeric DNA G-quadruplex, and the others are in substituted acridine bimolecular G-quadruplex complexes. Bridging water molecules form most of the hydrogen-bond contacts between ligands and DNA in the parallel G-quadruplex structures examined here. Clusters of structured water molecules play essential roles in mediating between ligand side chain groups/chromophore core and G-quadruplex. These clusters tend to be conserved between complex and native G-quadruplex structures, suggesting that they more generally serve as platforms for ligand binding, and should be taken into account in docking and in silico studies.

TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2–TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein–DNA structures, and monitoring of DNA–DNA and DNA–RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA–dsDNA, dsDNA–ssDNA and dsDNA–ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

Emerging Accessibility Patterns in Long Telomeric Overhangs

We present single molecule experimental and computational modeling studies investigating the accessibility and folding landscape of human telomeric overhangs of physiologically relevant lengths. The overhangs contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate 1-7 tandem G-quadruplex (GQ) structures. Using FRET-PAINT, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to unfolded sites. We report accessibility patterns that periodically change with overhang length and provide insights about the underlying folding frustration. Overhangs that have 4n G-Tracts, (12, 16...), demonstrate maximum frustration, while those with 4n+2 G-Tracts, (14, 18...), have minimal frustration. We also developed a computational model that suggests positive folding cooperativity between neighboring GQs is required for persistence of such patterns. Our experimental and computational studies suggest lower folding stability at the junction between single and double stranded telomeric DNA, which has implications for Shelterin complex formation.

MRE11 and UBR5 Co-Operate to Suppress RNF168-Mediated Fusion of Dysfunctional Telomeres

TRF2 is part of the shelterin complex that hides telomeric DNA ends and prevents the activation of the cNHEJ pathway that can lead to chromosomal fusion. TRF2, however, also actively suppresses the cNHEJ pathway by recruiting two proteins, MRE11 and UBR5. MRE11 binds BRCC3, which in turn deubiquitinates γH2AX deposited at exposed telomeric DNA ends and limits RNF168 recruitment to the telomere. UBR5, in contrast directly ubiquitinates and destroys RNF168. The loss of telomeric RNF168 in turn blocks the subsequent recruitment of 53BP1 and prevents the cNHEJ-mediated fusion of chromosomes with exposed telomeric DNA ends. Although MRE11 and UBR5 are both involved in the control of telomeric RNF168 levels and the chromosome fusion process, their relative contributions have not been directly addressed. To do so we genetically suppressed MRE11 and UBR5 alone or in combination in glioma cell lines which we previously showed contained dysfunctional telomeres that were dependent on TRF2 for suppression of telomeric fusion and monitored the effects on events associated with telomere fusion. We here show that while suppression of either MRE11 or UBR5 alone had minimal effects on RNF168 telomeric accumulation, 53BP1 recruitment, and telomeric fusion, their combined suppression led to significant increases in RNF168 and 53BP1 telomeric recruitment and telomeric fusion and eventually cell death, all of which were reversible by suppression of RNF168 itself. These results show that MRE11 and UBR5 co-operate to suppress fusion at dysfunctional telomeres.

Roles for the 8-Oxoguanine DNA Repair System in Protecting Telomeres From Oxidative Stress

Telomeres are protective nucleoprotein structures that cap linear chromosome ends and safeguard genome stability. Progressive telomere shortening at each somatic cell division eventually leads to critically short and dysfunctional telomeres, which can contribute to either cellular senescence and aging, or tumorigenesis. Human reproductive cells, some stem cells, and most cancer cells, express the enzyme telomerase to restore telomeric DNA. Numerous studies have shown that oxidative stress caused by excess reactive oxygen species is associated with accelerated telomere shortening and dysfunction. Telomeric repeat sequences are remarkably susceptible to oxidative damage and are preferred sites for the production of the mutagenic base lesion 8-oxoguanine, which can alter telomere length homeostasis and integrity. Therefore, knowledge of the repair pathways involved in the processing of 8-oxoguanine at telomeres is important for advancing understanding of the pathogenesis of degenerative diseases and cancer associated with telomere instability. The highly conserved guanine oxidation (GO) system involves three specialized enzymes that initiate distinct pathways to specifically mitigate the adverse effects of 8-oxoguanine. Here we introduce the GO system and review the studies focused on investigating how telomeric 8-oxoguanine processing affects telomere integrity and overall genome stability. We also discuss newly developed technologies that target oxidative damage selectively to telomeres to investigate roles for the GO system in telomere stability.

Zipper head mechanism of telomere synthesis by human telomerase

Telomerase, a multi-subunit ribonucleoprotein complex, is a unique reverse transcriptase that catalyzes the processive addition of a repeat sequence to extend the telomere end using a short fragment of its own RNA component as the template. Despite recent structural characterizations of human and Tetrahymena telomerase, it is still a mystery how telomerase repeatedly uses its RNA template to synthesize telomeric DNA. Here, we report the cryo-EM structure of human telomerase holoenzyme bound with telomeric DNA at resolutions of 3.5 Å and 3.9 Å for the catalytic core and biogenesis module, respectively. The structure reveals that a leucine residue Leu980 in telomerase reverse transcriptase (TERT) catalytic subunit functions as a zipper head to limit the length of the short primer–template duplex in the active center. Moreover, our structural and computational analyses suggest that TERT and telomerase RNA (hTR) are organized to harbor a preformed active site that can accommodate short primer–template duplex substrates for catalysis. Furthermore, our findings unveil a double-fingers architecture in TERT that ensures nucleotide addition processivity of human telomerase. We propose that the zipper head Leu980 is a structural determinant for the sequence-based pausing signal of DNA synthesis that coincides with the RNA element-based physical template boundary. Functional analyses unveil that the non-glycine zipper head plays an essential role in both telomerase repeat addition processivity and telomere length homeostasis. In addition, we also demonstrate that this zipper head mechanism is conserved in all eukaryotic telomerases. Together, our study provides an integrated model for telomerase-mediated telomere synthesis.

Gain-of-Function Mutations in RPA1 Cause a Syndrome with Short Telomeres and Somatic Genetic Rescue

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify three germline heterozygous missense variants in RPA1 gene in four unrelated probands presenting with short telomeres and varying clinical features of TBD/STS including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function while RPA1T270A has binding properties similar to wild type protein. To study the mutational effect in a cellular system, we used CRISPR/Cas9 to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patient with RPA1E240K, we discovered somatic genetic rescue (SGR) in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the two SGR events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.