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BRCA1 binds TERRA RNA and suppresses R-Loop-based telomeric DNA damage.

ABSTRACT: R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.

SUBMITTER: Vohhodina J

PROVIDER: S-EPMC8192922 | biostudies-literature |

REPOSITORIES: biostudies-literature

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BRCA1 maintains telomere integrity through suppression of TERRA RNA and TERRA R-loop-induced DNA damage

Project description:Data in support of Vohhodina et al. "BRCA1 maintains telomere integrity through suppression of TERRA RNA and TERRA R-loop-induced DNA damage"

2021-04-21 | MSV000087276 | MassIVE

Increased amounts and stability of telomeric repeat-containing RNA (TERRA) following DNA damage induced by etoposide.

Project description:Telomeric repeat-containing RNAs (TERRAs) are long noncoding RNAs transcribed from subtelomeres toward telomeric repeat tracts, which have been implicated in telomere protection and heterochromatin formation. Genotoxic stress leads to upregulation of TERRAs. However, the mechanism of DNA damage-mediated TERRA induction remains elusive. Here, we treated HeLa cells with etoposide, a DNA double-strand break-generating agent, for various times and monitored the levels of TERRAs. Etoposide treatment led to a gradual time-dependent increase in TERRAs. Etoposide-mediated induction was evident in many TERRAs arising from various chromosome loci, including 20q and XpYp. Chromatin immunoprecipitation assays revealed no significant changes in the occupancy of RNA polymerase II at telomeres upon etoposide treatment. Interestingly, TERRAs arising from 20q, XpYp, 10q, and 13q degraded at slower rates in cells treated with etoposide, while degradation rates of TERRAs from many loci tested were nearly identical in both etoposide- and mock-treated cells. Telomere damage occurred from early time points of etoposide treatment, but telomere lengths and abundance of telomeric repeat-binding factor 2 (TRF2) at telomeres remained unchanged. In summary, etoposide treatment led to telomere damage and TERRA accumulation, but telomere lengths and TRF2-mediated telomere integrity were maintained. Etoposide-mediated TERRA accumulation could be attributed partly to RNA stabilization. These findings may provide insight into the post-transcriptional regulation of TERRAs in response to DNA damage.

| S-EPMC6874320 | biostudies-literature

Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids.

Project description:Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription.

| S-EPMC5449629 | biostudies-literature

A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex.

Project description:DNA telomeric repeats in mammalian cells are transcribed to guanine-rich RNA sequences, which adopt parallel-stranded G-quadruplexes with a propeller-like fold. The successful crystallization and structure analysis of a bimolecular human telomeric RNA G-quadruplex, folded into the same crystalline environment as an equivalent DNA oligonucleotide sequence, is reported here. The structural basis of the increased stability of RNA telomeric quadruplexes over DNA ones and their preference for parallel topologies is described here. Our findings suggest that the 2'-OH hydroxyl groups in the RNA quadruplex play a significant role in redefining hydration structure in the grooves and the hydrogen bonding networks. The preference for specific nucleotides to populate the C3'-endo sugar pucker domain is accommodated by alterations in the phosphate backbone, which leads to greater stability through enhanced hydrogen bonding networks. Molecular dynamics simulations on the DNA and RNA quadruplexes are consistent with these findings. The computations, based on the native crystal structure, provide an explanation for RNA G-quadruplex ligand binding selectivity for a group of naphthalene diimide ligands as compared to the DNA G-quadruplex.

| S-EPMC2938214 | biostudies-literature

An R-loop-initiated CSB-RAD52-POLD3 pathway suppresses ROS-induced telomeric DNA breaks.

Project description:Reactive oxygen species (ROS) inflict multiple types of lesions in DNA, threatening genomic integrity. How cells respond to ROS-induced DNA damage at telomeres is still largely unknown. Here, we show that ROS-induced DNA damage at telomeres triggers R-loop accumulation in a TERRA- and TRF2-dependent manner. Both ROS-induced single- and double-strand DNA breaks (SSBs and DSBs) contribute to R-loop induction, promoting the localization of CSB and RAD52 to damaged telomeres. RAD52 is recruited to telomeric R-loops through its interactions with both CSB and DNA:RNA hybrids. Both CSB and RAD52 are required for the efficient repair of ROS-induced telomeric DSBs. The function of RAD52 in telomere repair is dependent on its ability to bind and recruit POLD3, a protein critical for break-induced DNA replication (BIR). Thus, ROS-induced telomeric R-loops promote repair of telomeric DSBs through CSB-RAD52-POLD3-mediated BIR, a previously unknown pathway protecting telomeres from ROS. ROS-induced telomeric SSBs may not only give rise to DSBs indirectly, but also promote DSB repair by inducing R-loops, revealing an unexpected interplay between distinct ROS-induced DNA lesions.

| S-EPMC7026659 | biostudies-literature

Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes.

Project description:Telomeric repeat-containing RNA (TERRA) has been identified as a telomere-associated regulator of chromosome end protection. Here, we report that TERRA can also be found in extracellular fractions that stimulate innate immune signaling. We identified extracellular forms of TERRA in mouse tumor and embryonic brain tissue, as well as in human tissue culture cell lines using RNA in situ hybridization. RNA-seq analyses revealed TERRA to be among the most highly represented transcripts in extracellular fractions derived from both normal and cancer patient blood plasma. Cell-free TERRA (cfTERRA) could be isolated from the exosome fractions derived from human lymphoblastoid cell line (LCL) culture media. cfTERRA is a shorter form (?200 nt) of cellular TERRA and copurifies with CD63- and CD83-positive exosome vesicles that could be visualized by cyro-electron microscopy. These fractions were also enriched for histone proteins that physically associate with TERRA in extracellular ChIP assays. Incubation of cfTERRA-containing exosomes with peripheral blood mononuclear cells stimulated transcription of several inflammatory cytokine genes, including TNF?, IL6, and C-X-C chemokine 10 (CXCL10) Exosomes engineered with elevated TERRA or liposomes with synthetic TERRA further stimulated inflammatory cytokines, suggesting that exosome-associated TERRA augments innate immune signaling. These findings imply a previously unidentified extrinsic function for TERRA and a mechanism of communication between telomeres and innate immune signals in tissue and tumor microenvironments.

| S-EPMC4655533 | biostudies-literature

RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells.

Project description:A fraction of cancer cells maintain telomeres through the telomerase-independent, 'Alternative Lengthening of Telomeres' (ALT) pathway. ALT relies on homologous recombination (HR) between telomeric sequences; yet, what makes ALT telomeres recombinogenic remains unclear. Here we show that the RNA endonuclease RNaseH1 regulates the levels of RNA-DNA hybrids between telomeric DNA and the long noncoding RNA TERRA, and is a key mediator of telomere maintenance in ALT cells. RNaseH1 associated to telomeres specifically in ALT cells and its depletion led to telomeric hybrid accumulation, exposure of single-stranded telomeric DNA, activation of replication protein A at telomeres and abrupt telomere excision. Conversely, overexpression of RNaseH1 weakened the recombinogenic nature of ALT telomeres and led to telomere shortening. Altering cellular RNaseH1 levels did not perturb telomere homoeostasis in telomerase-positive cells. RNaseH1 maintains regulated levels of telomeric RNA-DNA hybrids at ALT telomeres to trigger HR without compromising telomere integrity too severely.

| S-EPMC4218956 | biostudies-literature

TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA.

Project description:Maintenance of telomeres requires both DNA replication and telomere 'capping' by shelterin. These two processes use two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the re-accumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA and POT1 act in concert to displace RPA from telomeric ssDNA after DNA replication, and promote telomere capping to preserve genomic integrity.

| S-EPMC3078637 | biostudies-literature

BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair.

Project description:The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites.

| S-EPMC4351672 | biostudies-literature

Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation.

Project description:The DNA-damage response (DDR) arrests cell-cycle progression until damage is removed. DNA-damage-induced cellular senescence is associated with persistent DDR. The molecular bases that distinguish transient from persistent DDR are unknown. Here we show that a large fraction of exogenously induced persistent DDR markers is associated with telomeric DNA in cultured cells and mammalian tissues. In yeast, a chromosomal DNA double-strand break next to a telomeric sequence resists repair and impairs DNA ligase 4 recruitment. In mammalian cells, ectopic localization of telomeric factor TRF2 next to a double-strand break induces persistent DNA damage and DDR. Linear, but not circular, telomeric DNA or scrambled DNA induces a prolonged checkpoint in normal cells. In terminally differentiated tissues of old primates, DDR markers accumulate at telomeres that are not critically short. We propose that linear genomes are not uniformly reparable and that telomeric DNA tracts, if damaged, are irreparable and trigger persistent DDR and cellular senescence.

| S-EPMC3717580 | biostudies-literature

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