Author Correction to: Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage

An amendment to this paper has been published and can be accessed via the original article.</p

Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage

BACKGROUND: The main function of telomerase is at the telomeres but under adverse conditions telomerase can bind to internal regions causing deleterious effects as observed in cancer cells. RESULTS: By mapping the global occupancy of the catalytic subunit of telomerase (Est2) in the budding yeast Saccharomyces cerevisiae, we reveal that it binds to multiple guanine-rich genomic loci, which we termed “non-telomeric binding sites” (NTBS). We characterize Est2 binding to NTBS. Contrary to telomeres, Est2 binds to NTBS in G1 and G2 phase independently of Est1 and Est3. The absence of Est1 and Est3 renders telomerase inactive at NTBS. However, upon global DNA damage, Est1 and Est3 join Est2 at NTBS and telomere addition can be observed indicating that Est2 occupancy marks NTBS regions as particular risks for genome stability. CONCLUSIONS: Our results provide a novel model of telomerase regulation in the cell cycle using internal regions as “parking spots” of Est2 but marking them as hotspots for telomere addition. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-021-01167-1

DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential.

Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy.The Balasubramanian laboratory is core-funded by Cancer Research UK (C14303/A17197) and further supported by a Cancer Research UK programme grant (C9681/A18618). S.B. is a Wellcome Trust Senior Investigator (099232/Z/12/Z)

Rudimentary G-Quadruplex-Based Telomere Capping In Saccharomyces Cerevisiae

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA-stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3\u27 overhang inhibits 5\u27-\u3e 3\u27 resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo

G-Quadruplex DNA Sequences Are Evolutionarily Conserved and Associated with Distinct Genomic Features in Saccharomyces cerevisiae

G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint

G-quadruplex formation at the 3′ end of telomere DNA inhibits its extension by telomerase, polymerase and unwinding by helicase

Telomere G-quadruplex is emerging as a promising anti-cancer target due to its inhibition to telomerase, an enzyme expressed in more than 85% tumors. Telomerase-mediated telomere extension and some other reactions require a free 3′ telomere end in single-stranded form. G-quadruplex formation near the 3′ end of telomere DNA can leave a 3′ single-stranded tail of various sizes. How these terminal structures affect reactions at telomere end is not clear. In this work, we studied the 3′ tail size-dependence of telomere extension by either telomerase or the alternative lengthening of telomere (ALT) mechanism as well as telomere G-quadruplex unwinding. We show that these reactions require a minimal tail of 8, 12 and 6 nt, respectively. Since we have shown that G-quadruplex tends to form at the farthest 3′ distal end of telomere DNA leaving a tail of no more than 5 nt, these results imply that G-quadruplex formation may play a role in regulating reactions at the telomere ends and, as a result, serve as effective drug target for intervening telomere function

ATRX dysfunction Induces replication defects in primary mouse cells

The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells

Genome-Wide Analyses of Recombination Prone Regions Predict Role of DNA Structural Motif in Recombination

HapMap findings reveal surprisingly asymmetric distribution of recombinogenic regions. Short recombinogenic regions (hotspots) are interspersed between large relatively non-recombinogenic regions. This raises the interesting possibility of DNA sequence and/or other cis- elements as determinants of recombination. We hypothesized the involvement of non-canonical sequences that can result in local non-B DNA structures and tested this using the G-quadruplex DNA as a model. G-quadruplex or G4 DNA is a unique form of four-stranded non-B DNA structure that engages certain G-rich sequences, presence of such motifs has been noted within telomeres. In support of this hypothesis, genome-wide computational analyses presented here reveal enrichment of potential G4 (PG4) DNA forming sequences within 25618 human hotspots relative to 9290 coldspots (p<0.0001). Furthermore, co-occurrence of PG4 DNA within several short sequence elements that are associated with recombinogenic regions was found to be significantly more than randomly expected. Interestingly, analyses of more than 50 DNA binding factors revealed that co-occurrence of PG4 DNA with target DNA binding sites of transcription factors c-Rel, NF-kappa B (p50 and p65) and Evi-1 was significantly enriched in recombination-prone regions. These observations support involvement of G4 DNA in recombination, predicting a functional model that is consistent with duplex-strand separation induced by formation of G4 motifs in supercoiled DNA and/or when assisted by other cellular factors

G-quadruplex structures mark human regulatory chromatin

G-quadruplex (G4) structural motifs have been linked to transcription, replication and genome instability and are implicated in cancer and other diseases. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context. Herein we address this through the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5' UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as $\textit{MYC}$. Strikingly, $\textit{de novo}$ and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor-induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.European Molecular Biology Organization (EMBO Long-Term Fellowship), University of Cambridge, Cancer Research UK (Grant ID: C14303/A17197), Wellcome Trust (Grant ID: 099232/z/12/z