Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein.

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

Platinum and PARP Inhibitor Resistance Due to Overexpression of MicroRNA-622 in BRCA1-Mutant Ovarian Cancer.

High-grade serous ovarian carcinomas (HGSOCs) with BRCA1/2 mutations exhibit improved outcome and sensitivity to double-strand DNA break (DSB)-inducing agents (i.e., platinum and poly(ADP-ribose) polymerase inhibitors [PARPis]) due to an underlying defect in homologous recombination (HR). However, resistance to platinum and PARPis represents a significant barrier to the long-term survival of these patients. Although BRCA1/2-reversion mutations are a clinically validated resistance mechanism, they account for less than half of platinum-resistant BRCA1/2-mutated HGSOCs. We uncover a resistance mechanism by which a microRNA, miR-622, induces resistance to PARPis and platinum in BRCA1 mutant HGSOCs by targeting the Ku complex and restoring HR-mediated DSB repair. Physiologically, miR-622 inversely correlates with Ku expression during the cell cycle, suppressing non-homologous end-joining and facilitating HR-mediated DSB repair in S phase. Importantly, high expression of miR-622 in BRCA1-deficient HGSOCs is associated with worse outcome after platinum chemotherapy, indicating microRNA-mediated resistance through HR rescue.

Dyskeratosis congenita mutations in dyskerin SUMOylation consensus sites lead to impaired telomerase RNA accumulation and telomere defects.

Mutations in the dyskerin gene (DKC1) cause X-linked dyskeratosis congenita (DC), a rare and fatal premature aging syndrome characterized by defective telomere maintenance. Dyskerin is a highly conserved nucleolar protein, and a component of the human telomerase complex that is essential for human telomerase RNA (hTR) stability. However, its regulation remains poorly understood. Here, we report that dyskerin can be modified by small ubiquitin-like modifiers (SUMOs). We find that human DC-causing mutations in highly conserved dyskerin SUMOylation consensus sites lead to impaired hTR accumulation, telomerase activity and telomere maintenance. Finally, we show that modification of dyskerin by SUMOylation is required for its stability. Our findings provide the first evidence that dyskerin stability is regulated by SUMOylation and that mutations altering dyskerin SUMOylation can lead to defects in telomere maintenance that are characteristics of DC.

Charity begins at home: non-coding RNA functions in DNA repair.

During the past decade, evolutionarily conserved microRNAs (miRNAs) have been characterized as regulators of almost every cellular process and signalling pathway. There is now emerging evidence that this new class of regulators also impinges on the DNA damage response (DDR). Both miRNAs and other small non-coding RNAs (ncRNAs) are induced at DNA breaks and mediate the repair process. These intriguing observations raise the possibility that crosstalk between ncRNAs and the DDR might provide a means of efficient and accurate DNA repair and facilitate the maintenance of genomic stability.

Telomeric recombination induced by dysfunctional telomeres.

Telomere maintenance is essential for cellular immortality, and most cancer cells maintain their telomeres through the enzyme telomerase. Telomeres and telomerase represent promising anticancer targets. However, 15% of cancer cells maintain their telomeres through alternative recombination-based mechanisms, and previous analyses showed that recombination-based telomere maintenance can be activated after telomerase inhibition. We determined whether telomeric recombination can also be promoted by telomere dysfunction. We report for the first time that telomeric recombination can be induced in human telomerase-positive cancer cells with dysfunctional telomeres.

Growth defects in mouse telomerase RNA-deficient cells expressing a template-mutated mouse telomerase RNA.

Cellular viability requires telomere maintenance, which, in mammals, is mainly mediated by the reverse transcriptase telomerase. Telomerase core components are a catalytic subunit TERT and an RNA subunit TR (hTR in humans, mTR in mouse) that carries the template to generate telomeres de novo. Telomere dysfunction can lead to senescence or apoptosis and impairs the continued growth of immortal cancerous cell lines. The introduction of a template-mutated hTR in telomerase-positive and telomerase-negative human cell lines results in dramatic growth defects. No study has addressed the consequences of expressing a template-mutated mTR in mouse immortal cell lines. Therefore, we analyzed the effects of long-term expression of a template-mutated mTR in the telomerase-positive and telomerase-negative murine cell lines CB17 and DKO301, respectively. Whereas the CB17 clones expressing the template-mutated mTR did not demonstrate any growth impairment, many of the DKO301 clones expressing the template-mutated mTR underwent growth and cell cycle defects and eventual cell death. These results suggest that in the absence of wild-type telomerase, the expression of the template-mutated mTR likely perturbs telomere function, leading to decreased cellular viability. Furthermore, whereas the expression of template-mutated hTR in telomerase-negative human cell lines leads to immediate cellular toxicity, the expression of the template-mutated mTR in the telomerase-negative mouse cell line did not.

Telomerase inhibition in a mouse cell line with long telomeres leads to rapid telomerase reactivation.

The indefinite growth of cancer cells requires telomere maintenance, which, in the majority of mammalian cancers is mediated via the enzyme telomerase. The core components of telomerase are a catalytic reverse transcriptase (hTERT in human, mTERT in mouse) and an RNA (TR) that contains the template for the replenishment of telomeres. Fundamental differences in human and mouse telomerase and telomere biology should be considered when using mouse models for the study of human cancers. The responses to telomerase inhibition by the expression of a catalytically-inactive dominant-negative mutant of hTERT (hTERT-DN) vary in human cells with different telomere lengths. Only one similar study has been performed in a mouse cell line with short telomeres (RenCa, 7 kb). Thus, we asked whether the responses to telomerase inhibition are also telomere-length dependent in mouse cells by analyzing long-term stable expression of mTERT-DN in the CB17 cell line (telomere length, 11 kb). A brief initial telomerase inhibition was insufficient to mediate telomere shortening and led to extremely rapid telomerase reactivation due to an increase in the level of expression of the endogenous mTERT. Thus, mouse cells, in contrast to human cells may not tolerate telomerase inhibition by introduction of mTERT-DN, independently of telomere length.