Recruitment Of BRCA1 Research Articles

Titanium Activates the DNA Damage Response Pathway in Oral Epithelial Cells: A Pilot Study.

To evaluate the effect of titanium (Ti) particles on oral epithelial cell homeostasis and the potential of dental implants to release Ti debris upon insertion. Dental implants with varying surface treatments were employed to determine the feasibility of particle release during implant placement as well as the impact of free Ti debris on oral epithelial cells. Ti particles derived from implant surfaces were isolated and cultured in direct contact with normal oral epithelial cells for 48 hours. Further, cells were fixed and processed for immunofluorescence assay to detect the activation of the DNA damage response (DDR) using CHK2 and BRCA1 molecular markers. Positive cells demonstrating DNA damage were quantified and statistically analyzed. Ti particles derived from implants containing phosphate-enriched titanium oxide (PETO), fluoride-modified (FM), and grit-blasted (GB) surface treatments were able to activate CHK2 and trigger the recruitment of BRCA1 in oral epithelial cells. Also, implants with GB surfaces were able to release Ti particles upon implant placement. The results indicate that Ti debris may be detached from the implant surface upon placement. Also, free Ti particles can trigger DDR signaling in oral epithelial cells. These findings suggest that Ti particles/debris released into a surgical wound may contribute to the disruption of epithelial homeostasis, and potentially compromise the oral epithelial barrier.

FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

In the mammalian genome, certain genomic loci/regions pose greater challenges to the DNA replication machinery (i.e., the replisome) than others. Such known genomic loci/regions include centromeres, common fragile sites, subtelomeres, and telomeres. However, the detailed mechanism of how mammalian cells cope with the replication stress at these loci/regions is largely unknown. Here we show that depletion of FANCM, or of one of its obligatory binding partners, FAAP24, MHF1, and MHF2, induces replication stress primarily at the telomeres of cells that use the alternative lengthening of telomeres (ALT) pathway as their telomere maintenance mechanism. Using the telomere-specific single-molecule analysis of replicated DNA technique, we found that depletion of FANCM dramatically reduces the replication efficiency at ALT telomeres. We further show that FANCM, BRCA1, and BLM are actively recruited to the ALT telomeres that are experiencing replication stress and that the recruitment of BRCA1 and BLM to these damaged telomeres is interdependent and is regulated by both ATR and Chk1. Mechanistically, we demonstrated that, in FANCM-depleted ALT cells, BRCA1 and BLM help to resolve the telomeric replication stress by stimulating DNA end resection and homologous recombination (HR). Consistent with their roles in resolving the replication stress induced by FANCM deficiency, simultaneous depletion of BLM and FANCM, or of BRCA1 and FANCM, leads to increased micronuclei formation and synthetic lethality in ALT cells. We propose that these synthetic lethal interactions can be explored for targeting the ALT cancers.

Nuclear Acetyl-CoA Production by ACLY Promotes Homologous Recombination

While maintaining the integrity of the genome and sustaining bioenergetics are both fundamental functions of the cell, potential crosstalk between metabolic and DNA repair pathways is poorly understood. Since histone acetylation plays important roles in DNA repair and is sensitive to the availability of acetyl coenzyme A (acetyl-CoA), we investigated a role for metabolic regulation of histone acetylation during the DNA damage response. In this study, we report that nuclear ATP-citrate lyase (ACLY) is phosphorylated at S455 downstream of ataxia telangiectasia mutated (ATM) and AKT following DNA damage. ACLY facilitates histone acetylation at double-strand break (DSB) sites, impairing 53BP1 localization and enabling BRCA1 recruitment and DNA repair by homologous recombination. ACLY phosphorylation and nuclear localization are necessary for its role inpromoting BRCA1 recruitment. Upon PARP inhibition, ACLY silencing promotes genomic instability and cell death. Thus, the spatial and temporal control of acetyl-CoA production by ACLY participates in the mechanism of DNA repair pathway choice.

Put Your Mark Where Your Damage Is: Acetyl-CoA Production by ACLY Promotes DNA Repair

In this issue of Molecular Cell, Sivanand etal. (2017) describe the importance for nuclear ACLY-mediated production of acetyl-CoA, which promotes histone acetylation, BRCA1 recruitment, and subsequent HR-mediated DNA repair in response to DNA damage, thus drawing a direct link between DNA repair and cellular metabolism.

The anaphase promoting complex impacts repair choice by protecting ubiquitin signalling at DNA damage sites

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). While HDR can only occur in S/G2, NHEJ can happen in all cell cycle phases (except mitosis). How then is the repair choice made in S/G2 cells? Here we provide evidence demonstrating that APCCdh1 plays a critical role in choosing the repair pathways in S/G2 cells. Our results suggest that the default for all DSBs is to recruit 53BP1 and RIF1. BRCA1 is blocked from being recruited to broken ends because its recruitment signal, K63-linked poly-ubiquitin chains on histones, is actively destroyed by the deubiquitinating enzyme USP1. We show that the removal of USP1 depends on APCCdh1 and requires Chk1 activation known to be catalysed by ssDNA-RPA-ATR signalling at the ends designated for HDR, linking the status of end processing to RIF1 or BRCA1 recruitment.

RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage

Ubiquitin modification of proteins plays pivotal roles in the cellular response to DNA damage. Given the complexity of ubiquitin conjugation due to the formation of poly-conjugates of different linkages, functional roles of linkage-specific ubiquitin modification at DNA damage sites are largely unclear. We identify that Lys11-linkage ubiquitin modification occurs at DNA damage sites in an ATM-dependent manner, and ubiquitin-modifying enzymes, including Ube2S E2-conjugating enzyme and RNF8 E3 ligase, are responsible for the assembly of Lys11-linkage conjugates on damaged chromatin, including histone H2A/H2AX. We show that RNF8- and Ube2S-dependent Lys11-linkage ubiquitin conjugation plays an important role in regulating DNA damage-induced transcriptional silencing, distinct from the role of Lys63-linkage ubiquitin in the recruitment of DNA damage repair proteins 53BP1 and BRCA1. Thus, our study highlights the importance of linkage-specific ubiquitination at DNA damage sites, and it reveals that Lys11-linkage ubiquitin modification plays a crucial role in the DNA damage response.

Abstract IA20: Phosphorylation of BRCA1 by CHK2 mediates resection activity and recruitment of BRCA2

Abstract There is genetic evidence that Chk2, BRCA1 and BRCA2 are functioning in the same pathway of DNA repair, but the links between the proteins are poorly understood. We have previously shown that BRCA1's regulation of homologous recombination (HR) is dependent on Chk2-mediated phosphorylation of BRCA1 at serine 988 (BRCA1-S988). We also found that BRCA1 regulates BRCA2 recruitment in response to DNA damage, which is also dependent on the phosphorylation of BRCA1 at Serine-988. Furthermore, in HCT116/Chk2-/- cells and in MCF7 cells with depleted Chk2, the same effects on HR and BRCA2 recruitment has been found as found in cells expressing the BRCA1-S988A protein. However, in both cases, BRCA1 is recruited to chromatin and into nuclear foci, so the effect of Chk2 is not affecting the recruitment of BRCA1. BRCA1-S988A shows a defect in RPA phosphorylation in response to replication stress and DNA damage. After global DNA damage with X-rays, MRE11 foci form at double-strand breaks, and in S and G2 phases of the cell cycle, an association with CTIP is observed transiently. In the presence of BRCA1-S988A, MRE11 co-localized with CTIP persists for much longer, and results in reduced single-stranded DNA bound by RPA. In response to a site-specific DSB induced by I-SceI nuclease, BRCA1-S988A can localize to the vicinity of the break, but not directly at the broken end, as observed by chromatin immuno-precipitation. A physical assay of resection, which distinguishes single-stranded DNA from double-stranded DNA by its susceptibility to restriction enzyme digestion, shows that BRCA1-S988A expressing cells have an initiation of resection, but a failure to extend resection up to 2 kb observed in wild-type cells. The recruitment of Exo1 is being investigated. Therefore, these results demonstrate that BRCA1-S988 plays key role in regulating the extent of resection, which in turn impedes the recruitment of BRCA2 to sites of resected DSB. Chk2 inhibition may exacerbate the defect in HR, particularly in cells with a partial or mild HR-deficiency, which is found quite commonly in human tumor cells, suggesting that combination of Chk2 and PARP inhibitors may be a useful combination DNA repair based therapy. Citation Format: Simon N. Powell. Phosphorylation of BRCA1 by CHK2 mediates resection activity and recruitment of BRCA2 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA20.

Ataxin‐3 consolidates the MDC1‐dependent DNA double‐strand break response by counteracting the SUMO‐targeted ubiquitin ligase RNF4

The SUMO‐targeted ubiquitin ligase RNF4 functions at the crossroads of the SUMO and ubiquitin systems. Here, we report that the deubiquitylation enzyme (DUB) ataxin‐3 counteracts RNF4 activity during the DNA double‐strand break (DSB) response. We find that ataxin‐3 negatively regulates ubiquitylation of the checkpoint mediator MDC1, a known RNF4 substrate. Loss of ataxin‐3 markedly decreases the chromatin dwell time of MDC1 at DSBs, which can be fully reversed by co‐depletion of RNF4. Ataxin‐3 is recruited to DSBs in a SUMOylation‐dependent fashion, and in vitro it directly interacts with and is stimulated by recombinant SUMO, defining a SUMO‐dependent mechanism for DUB activity toward MDC1. Loss of ataxin‐3 results in reduced DNA damage‐induced ubiquitylation due to impaired MDC1‐dependent recruitment of the ubiquitin ligases RNF8 and RNF168, and reduced recruitment of 53BP1 and BRCA1. Finally, ataxin‐3 is required for efficient MDC1‐dependent DSB repair by non‐homologous end‐joining and homologous recombination. Consequently, loss of ataxin‐3 sensitizes cells to ionizing radiation and poly(ADP‐ribose) polymerase inhibitor. We propose that the opposing activities of RNF4 and ataxin‐3 consolidate robust MDC1‐dependent signaling and repair of DSBs.

MCL-1 Depletion Impairs DNA Double-Strand Break Repair and Reinitiation of Stalled DNA Replication Forks.

Myeloid cell leukemia 1 (MCL-1) is a prosurvival BCL-2 protein family member highly expressed in hematopoietic stem cells (HSCs) and regulated by growth factor signals that manifest antiapoptotic activity. Here we report that depletion of MCL-1 but not its isoform MCL-1S increases genomic instability and cell sensitivity to ionizing radiation (IR)-induced death. MCL-1 association with genomic DNA increased postirradiation, and the protein colocalized with 53BP1 foci. Postirradiation, MCL-1-depleted cells exhibited decreased γ-H2AX foci, decreased phosphorylation of ATR, and higher levels of residual 53BP1 and RIF1 foci, suggesting that DNA double-strand break (DSB) repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1-depleted cells had a reduced frequency of IR-induced BRCA1, RPA, and Rad51 focus formation, decreased DNA end resection, and decreased HR repair in the DR-GFP DSB repair model. Similarly, after HU induction of stalled replication forks in MCL-1-depleted cells, there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR-mediated resolution of collapsed forks. Therefore, the present data support a model whereby MCL-1 depletion increases 53BP1 and RIF1 colocalization at DSBs, which inhibits BRCA1 recruitment, and sensitizes cells to DSBs from IR or stalled replication forks that require HR for repair.

Germline missense pathogenic variants in the BRCA1 BRCT domain, p.Gly1706Glu and p.Ala1708Glu, increase cellular sensitivity to PARP inhibitor olaparib by a dominant negative effect.

BRCA1-deficient cells show defects in DNA repair and rely on other members of the DNA repair machinery, which makes them sensitive to PARP inhibitors (PARPi). Although carrying a germline pathogenic variant in BRCA1/2 is the best determinant of response to PARPi, a significant percentage of the patients do not show sensitivity and/or display increased toxicity to the agent. Considering previously suggested mutation-specific BRCA1 haploinsufficiency, we aimed to investigate whether there are any differences in cellular response to PARPi olaparib depending on the BRCA1 mutation type. Lymphoblastoid cell lines derived from carriers of missense pathogenic variants in the BRCA1 BRCT domain (c.5117G > A, p.Gly1706Glu and c.5123C > A, p.Ala1708Glu) showed higher sensitivity to olaparib than cells with truncating variants or wild types (WT). Response to olaparib depended on a basal PARP enzymatic activity, but did not correlate with PARP1 expression. Interestingly, cellular sensitivity to the agent was associated with the level of BRCA1 recruitment into γH2AX foci, being the lowest in cells with missense variants. Since these variants lead to partially stable protein mutants, we propose a model in which the mutant protein acts in a dominant negative manner on the WT BRCA1, impairing the recruitment of BRCA1 into DNA damage sites and, consequently, increasing cellular sensitivity to PARPi. Taken together, our results indicate that carriers of different BRCA1 mutations could benefit from olaparib in a distinct way and show different toxicities to the agent, which could be especially relevant for a potential future use of PARPi as prophylactic agents in BRCA1 mutation carriers.

INT6/EIF3E Controls the RNF8-Dependent Ubiquitylation Pathway and Facilitates DNA Double-Strand Break Repair in Human Cells.

Unrepaired DNA double-strand breaks (DSB) are the most destructive chromosomal lesions driving genomic instability, a core hallmark of cancer. Here, we identify the antioncogenic breast cancer factor INT6/EIF3E as an essential regulator of DSB repair that promotes homologous recombination (HR)-mediated repair and, to a lesser extent, nonhomologous end-joining repair. INT6 silencing impaired the accrual of the ubiquitin ligase RNF8 at DSBs and the formation of ubiquitin conjugates at DSB sites, especially Lys63-linked polyubiquitin chains, resulting in impaired recruitment of BRCA1, BRCA2, and RAD51, which are all involved in HR repair. In contrast, INT6 deficiency did not affect the accumulation of RNF168, 53BP1, or RPA at DSBs. In INT6-silenced cells, there was also an alteration in DNA damage-induced localization of MDC1, a key target for ATM phosphorylation, which is a prerequisite for RNF8 recruitment. The attenuated DNA damage localization of RNF8 resulting from INT6 depletion could be attributed to the defective retention of ATM previously reported by us. Our findings deepen insights into how INT6 protects against breast cancer by showing how it functions in DSB repair, with potential clinical implications for cancer therapy. Cancer Res; 76(20); 6054-65. ©2016 AACR.

A role for the p53 tumour suppressor in regulating the balance between homologous recombination and non-homologous end joining.

Loss of p53, a transcription factor activated by cellular stress, is a frequent event in cancer. The role of p53 in tumour suppression is largely attributed to cell fate decisions. Here, we provide evidence supporting a novel role for p53 in the regulation of DNA double-strand break (DSB) repair pathway choice. 53BP1, another tumour suppressor, was initially identified as p53 Binding Protein 1, and has been shown to inhibit DNA end resection, thereby stimulating non-homologous end joining (NHEJ). Yet another tumour suppressor, BRCA1, reciprocally promotes end resection and homologous recombination (HR). Here, we show that in both human and mouse cells, the absence of p53 results in impaired 53BP1 focal recruitment to sites of DNA damage induced by ionizing radiation. This effect is largely independent of cell cycle phase and the extent of DNA damage. In p53-deficient cells, diminished localization of 53BP1 is accompanied by a reciprocal increase in BRCA1 recruitment to DSBs. Consistent with these findings, we demonstrate that DSB repair via NHEJ is abrogated, while repair via homology-directed repair (HDR) is stimulated. Overall, we propose that in addition to its role as an ‘effector’ protein in the DNA damage response, p53 plays a role in the regulation of DSB repair pathway choice.

Factors forming the BRCA1-A complex orchestrate BRCA1 recruitment to the sites of DNA damage.

Sustaining genomic integrity is essential for preventing onset of cancers. Therefore, human cells evolve to have refined biological pathways to defend genetic materials from various genomic insults. DNA damage response and DNA repair pathways essential for genome maintenance are accomplished by cooperative executions of multiple factors including breast cancer type 1 susceptibility protein (BRCA1). BRCA1 is initially identified as an altered gene in the hereditary breast cancer patients. Since then, tremendous efforts to understand the functions of BRAC1 reveal that BRCA1 is found in distinct complexes, including BRCA1-A, BRCA1-B, BRCA1-C, and the BRCA1/PALB2/BRCA2 complex, and plays diverse roles in a context-dependent manner. Among the complexes, BRCA1-A is critical for BRCA1 recruitment to the sites of DNA damage. Factors comprising the BRCA1-A include RAP80, CCDC98/Abraxas, BRCC36, BRCC45, BARD1, BRCA1, and MERIT40, a RAP80-associated factor. In this review, we summarize recent findings of the factors that form the BRCA1-A complex.

ABRAXAS (FAM175A) and Breast Cancer Susceptibility: No Evidence of Association in the Breast Cancer Family Registry.

Approximately half of the familial aggregation of breast cancer remains unexplained. This proportion is less for early-onset disease where familial aggregation is greater, suggesting that other susceptibility genes remain to be discovered. The majority of known breast cancer susceptibility genes are involved in the DNA double-strand break repair pathway. ABRAXAS is involved in this pathway and mutations in this gene impair BRCA1 recruitment to DNA damage foci and increase cell sensitivity to ionizing radiation. Moreover, a recurrent germline mutation was reported in Finnish high-risk breast cancer families. To determine if ABRAXAS could be a breast cancer susceptibility gene in other populations, we conducted a population-based case-control mutation screening study of the coding exons and exon/intron boundaries of ABRAXAS in the Breast Cancer Family Registry. In addition to the common variant p.Asp373Asn, sixteen distinct rare variants were identified. Although no significant difference in allele frequencies between cases and controls was observed for the identified variants, two variants, p.Gly39Val and p.Thr141Ile, were shown to diminish phosphorylation of gamma-H2AX in MCF7 human breast adenocarcinoma cells, an important biomarker of DNA double-strand breaks. Overall, likely damaging or neutral variants were evenly represented among cases and controls suggesting that rare variants in ABRAXAS may explain only a small proportion of hereditary breast cancer.

Combinatory effect of BRCA1 and HERC2 expression on outcome in advanced non-small-cell lung cancer.

BackgroundBRCA1 is a main component of homologous recombination and induces resistance to platinum in preclinical models. It has been studied as a potential predictive marker in lung cancer. Several proteins modulate the function of BRCA1. The E3 ubiquitin ligase HERC2 facilitates the assembly of the RNF8-UBC13 complex to recruit BRCA1 to DNA damage sites. The combined analysis of multiple components of the pathway leading to the recruitment of BRCA1 at DNA damage sites has the potentiality to improve the BRCA1 predictive model.MethodsWe retrospectively analyzed 71 paraffin-embedded tumor samples from advanced non-small-cell lung cancer patients treated with first-line platinum based chemotherapy and measured the mRNA expression levels of BRCA1, RNF8, UBC13 and HERC2 using real-time PCR. The mRNA expression was categorized using median value as cut-off point.ResultsThe median progression-free survival of all 71 patients was 7.2 months whereas the median overall survival of the study population was 10.7 months. Among patients with low BRCA1 expression, the median PFS was 7.4 months in the presence of low HERC2 levels and 5.9 months for patients expressing high HERC2 levels (p = 0.01). The median OS was 15.3 months for patients expressing low levels of both genes and 7.4 months for those with low BRCA1 but high HERC2 (p = 0.008). The multivariate analysis showed that among patients with Eastern Cooperative Oncology Group performance status 0–1, the combined low expression of both BRCA1 and HERC2 clearly reduced the risk of progression (p = 0.03) and of death (p = 0.004).ConclusionsThese findings confirm the potentiality of integrated DNA repair components analysis in predicting the sensitivity to platinum in lung cancer. The study indicates a predictive role for HERC2 mRNA expression and paves the way for further refinement of the BRCA1 predictive model.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2339-5) contains supplementary material, which is available to authorized users.

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

Targeting the epigenetics of the DNA damage response in breast cancer.

Cancer is as much an epigenetic disease as it is a genetic disease, and epigenetic alterations in cancer often serve as potent surrogates for genetic mutations. Because the epigenetic factors involved in the DNA damage response are regulated by multiple elements, therapies to target specific components of the epigenetic machinery can be inefficient. In contrast, therapies aimed at inhibiting the methionine cycle can indirectly inhibit both DNA and protein methylation, and the wide variety of genes and pathways that are affected by these methylations make this global strategy very attractive. In the present study, we propose an adjuvant therapy that targets the epigenetics of the DNA damage response in breast cancer cells and that results in efficient apoptosis and a reduction in distant metastases in vivo. We observed that a combined therapy designed to uncouple adenosine metabolism using dipyridamole in the presence of a new synthetic antifolate, 3-O-(3,4,5-trimethoxybenzoyl)-(−)-catechin, simultaneously and efficiently blocked both the folic cycle and the methionine cycle in breast cancer cells and sensitized these cells to radiotherapy. The treatment impeded the recruitment of 53BP1 and BRCA1 to the chromatin regions flanking DNA double-strand breaks and thereby avoided the DNA damage responses in breast cancer cells that were exposed to ionizing radiation. In addition, this hypomethylating therapy was also efficient in reducing the self-renewal capability of breast cancer-initiating cells and induced reversion of mesenchymal phenotypes in breast cancer cells.

Small RNAs Recruit Chromatin-Modifying Enzymes MMSET and Tip60 to Reconfigure Damaged DNA upon Double-Strand Break and Facilitate Repair.

Recent reports have demonstrated that DNA double-strand break (DSB)-induced small RNAs (diRNA) play an important role in the DNA damage response (DDR). However, the molecular mechanism by which diRNAs regulate the DDR remains unclear. Here, we report that Dicer- and Drosha-dependent diRNAs function as guiding molecules to promote the recruitment of the methyltransferase MMSET (WHSC1) and the acetyltransferase Tip60 (KAT5) to the DSB, where local levels of histone H4 di- and tri-methylation at lysine 20 (H4K20me2, 3) and H4 acetylation at lysine 16 (H4K16Ac) were enhanced. These histone modification events resulted in an open, flexible chromatin configuration, as indicated by the increased release of histones γH2AX, H2AX, and H3 from damaged chromatin. Furthermore, we found that diRNA-associated AGO2 interacted with MMSET and Tip60 and that the diRNA binding and catalytic activities of AGO2 were dispensable for the interaction but required for the recruitment of MMSET and Tip60 to DSBs. Consequently, diRNA-mediated chromatin remodeling promoted DSB repair by enhancing the recruitment of Rad51 and BRCA1 to the DSB site. Taken together, our findings reveal an unexpected direct role for diRNAs in regulating chromatin remodeling to facilitate DSB repair, revealing a new layer of DDR regulation involving specialized RNA molecules. Cancer Res; 76(7); 1904-15. ©2016 AACR.

Abstract PR03: A lncRNA regulates DNA repair by homologous recombination

Abstract Introduction: Long non-coding RNAs (lncRNAs) have been shown to contribute to DNA damage response (DDR) by regulating gene expression. However, very little is known about the role that lncRNAs play in regulating DNA repair. In this study we characterized a lncRNA that regulates DNA repair by Homologous recombination (HR) by modulating recruitment of DNA repair factors at the sites of DNA damage. Experimental procedures: Control or DNA damage treated cells were processed for gene expression analysis, qPCR, live cell imaging, Immunochemistry, Western blotting, FISH and RNA Immunoprecipitation. Results: Using a genome-wide microarray screen we identified a novel ubiquitously expressed lncRNA, DDSR1 (DNA damage-sensitive RNA 1), which is induced upon DNA damage by several DNA double-strand break (DSB) agents. DDSR1 induction upon DNA damage is dependent on the ATM-NF-kB pathway. Loss of DDSR1 impairs cell proliferation, DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect upon DDSR1 knockdown is characterized by aberrant BRCA1 and RAP80 accumulation at DSB sites. DDSR1 interacts with hnRNPUL1, an RNA-binding protein involved in HR. Loss of hnRNPUL1 also results in aberrant BRCA1 and RAP80 recruitment at DSB sites. Our results indicate that DDSR1/hnRNPUL1 depletion results in HR inhibition due to reduced end resection caused by aberrant BRCA1 and RAP80 at DSBs. Conclusions: Our results reveal a previously unknown lncRNA involved in regulation of DDR by contributing DNA repair by HR. Our findings highlight the importance of DDSR1 in maintaining genome stability. Citation Format: Vivek Sharma, Simran Khurana, Nard Kubben, Kotb Abdelmohsen, Philipp Oberdoerffer, Myriam Gorospe, Tom Misteli. A lncRNA regulates DNA repair by homologous recombination. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR03.

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability.