LncRNA lnc-RI regulates homologous recombination repair of DNA double-strand breaks by stabilizing RAD51 mRNA as a competitive endogenous RNA
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DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. The current models of the mechanism of DSB repair are based on studies of DNA repair proteins. Long non-coding RNAs (lncRNAs) have recently emerged as new regulatory molecules, with diverse functions in biological processes. In the present study, we found that expression of the ionizing radiation-inducible lncRNA, lnc-RI, was correlate negatively with micronucleus frequencies in human peripheral blood lymphocytes. Knockdown of lnc-RI significantly increased spontaneous DSBs levels, which was confirmed to be associated with the decreased efficiency of homologous recombination (HR) repair of DSBs. The expression of RAD51, a key recombinase in the HR pathway, decreased sharply in lnc-RI-depressed cells. In a further investigation, we demonstrated that miR-193a-3p could bind with both lnc-RI and RAD51 mRNA and depressed the expression of lnc-RI and RAD51 mRNA. Lnc-RI acted as a competitive endogenous RNA (ceRNA) to stabilize RAD51 mRNA via competitive binding with miR-193a-3p and release of its inhibition of RAD51 expression. To our knowledge, this is the first study to demonstrate the role of lnc-RI in regulating HR repair of DSBs. The feedback loop established in the current study suggests that lnc-RI is critical for the maintenance of genomic stability.Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.
Non-homologous end joining
BRCA2 Protein
Homology directed repair
RAD52
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Mammalian RAD51 protein plays essential roles in DNA homologous recombination, DNA repair and cell proliferation. RAD51 activities are regulated by its associated proteins. It was previously reported that a ubiquitin-like protein, UBL1, associates with RAD51 in the yeast two-hybrid system. One function of UBL1 is to covalently conjugate with target proteins and thus modify their function. In the present study we found that non-conjugated UBL1 forms a complex with RAD51 and RAD52 proteins in human cells. Overexpression of UBL1 down-regulates DNA double-strand break-induced homologous recombination in CHO cells and reduces cellular resistance to ionizing radiation in HT1080 cells. With or without overexpressed UBL1, most homologous recombination products arise by gene conversion. However, overexpression of UBL1 reduces the fraction of bidirectional gene conversion tracts. Overexpression of a mutant UBL1 that is incapable of being conjugated retains the ability to inhibit homologous recombination. These results suggest a regulatory role for UBL1 in homologous recombination.
RAD52
Non-homologous end joining
Homology directed repair
FLP-FRT recombination
Replication protein A
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Abstract BRCA2 mediates homologous recombination (HR) via its direct interaction with Rad51. There is ample genetic and biological evidence that BRCA1 and BRCA2 are functioning in the same pathway of DNA repair, but the functional connection between the two proteins is poorly understood. Interestingly, a mutation of BRCA1 (S988A) localized normally to ionizing radiation induced nuclear foci (IRIF), but the BRCA2 protein could not be recruited to BRCA1 at the damage site. Phosphorylation of BRCA1 at the S988 site is carried out by Chk2, which was also required for BRCA2 to localize to sites of DNA damage and support homologous recombination. These observations suggest that CHK2-BRCA1-BRCA2-Rad51 function in a single pathway of homologous recombinational repair to prevent the development of breast cancer. Loss of function of BRCA1/BRCA2 is a tumor-specific finding, making the role of alternative repair pathways in these cells critical for their survival. We show that loss of Rad52 function is synthetic lethal with BRCA1, PALB2 or BRCA2 deficiency, whereas there is no impact on cell growth in wild-type cells. The frequency of both spontaneous and double-strand break-induced homologous recombination, plus ionizing radiation induced Rad51 foci, decreased when Rad52 was depleted in BRCA2-deficient cells, with no effect in BRCA2-complemented cells. The observation that Rad52-inactivation is synthetic lethal with BRCA2 makes Rad52 a target for therapy in these tumors. The BRCA1-BRCA2 pathway can be functionally inactivated in sporadic breast cancer, perhaps as frequently as 20% of cases, suggesting that the pool of patients with defects in homologous recombinational repair is larger than initially expected. The functional assessment of defects in homologous recombination was assessed by ionizing-radiation (ex-vivo exposure) induced Rad51 foci and confirmed by characteristic patterns of array comparative genomic hybridization. The inactivation of the BRCA1-BRCA2 pathway allows the use of specific therapies designed to exploit the defects in DNA repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN05-04.
RAD52
BRCA2 Protein
PALB2
Non-homologous end joining
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BRCA2 Protein
Non-allelic homologous recombination
FLP-FRT recombination
Non-homologous end joining
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Germline mutations in BRCA1 confer a high risk of breast and ovarian tumors. The role of BRCA1 in tumor suppression is not yet understood, but both transcription and repair functions have been ascribed. Evidence that BRCA1 is involved in DNA repair stems from its association with RAD51, a homolog of the yeast protein involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination. We report here that Brca1-deficient mouse embryonic stem cells have impaired repair of chromosomal DSBs by homologous recombination. The relative frequencies of homologous and nonhomologous DNA integration and DSB repair were also altered. The results demonstrate a caretaker role for BRCA1 in preserving genomic integrity by promoting homologous recombination and limiting mutagenic nonhomologous repair processes.
Homology directed repair
Non-homologous end joining
Ku80
Replication protein A
BRCA2 Protein
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Ubiquitylation has an important role as a signal transducer that regulates protein function, subcellular localization, or stability during the DNA damage response. In this study, we show that Ring domain E3 ubiquitin ligases RNF138 is recruited to DNA damage site quickly. And the recruitment is mediated through its Zinc finger domains. We further confirm that RNF138 is phosphorylated by ATM at Ser124. However, the phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicate that RAD51 assembly at DSB sites following irradiation is dramatically affected in RNF138-deficient cells. Hence, RNF138 is likely involved in regulating homologous recombination repair pathway. Consistently, efficiency of homologous recombination decreased observably in RNF138-depleted cells. In addition, RNF138-deficient cell is hypersensitive to DNA damage insults, such as IR and MMS. And the comet assay confirmed that RNF138 directly participated in DNA damage repair. Moreover, we find that RAD51D directly interacted with RNF138. And the recruitment of RAD51D to DNA damage site is delayed and unstable in RNF138-depleted cells. Taken together, these results suggest that RNF138 promotes the homologous recombination repair pathway.
Non-homologous end joining
Comet Assay
Homology directed repair
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RAD52
BRCA2 Protein
Non-homologous end joining
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Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals.
Non-homologous end joining
BRCA2 Protein
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Abstract The breast cancer susceptibility gene, BRCA2, mediates homologous recombination (HR) via its direct interaction with the Rad51 recombinase. There is ample genetic and biological evidence that BRCA1 and BRCA2 are functioning in the same pathway of DNA repair, but the functional connection between the two proteins is poorly understood. Interestingly, a mutation of BRCA1 (S988A) localized normally to ionizing radiation induced nuclear foci (IRIF), but the BRCA2 protein could not be recruited to BRCA1 at the damage site. Phosphorylation of BRCA1 at the S988 site is carried out by Chk2, which was also required for BRCA2 to localize to sites of DNA damage and support homologous recombination. These observations suggest that CHK2-BRCA1-BRCA2-Rad51 function in a single pathway of homologous recombinational repair to prevent the development of breast cancer. In familial breast cancer, one allele of BRCA1 or BRCA2 is germline mutant and the second allele is inactivated to allow tumor development. Thus, loss of function of BRCA1/BRCA2 is a tumor-specific finding, making the role of alternative repair pathways in these cells critical for their survival. We show that loss of Rad52 function is synthetic lethal with BRCA2 deficiency, whereas there is no impact on cell growth in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination, and ionizing radiation induced Rad51 foci, decreased by greater than 50%, when Rad52 was depleted in BRCA2-deficient cells, with no effect in BRCA2-complemented cells. Ionizing radiation-induced and S-phase associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double strand breaks and replication stalling independently of BRCA2. The observation that Rad52-inactivation is synthetic lethal with BRCA2 and that Rad52 acts independently of BRCA2 makes Rad52 a target for therapy in these tumors. Finally, we have evidence that the BRCA1-BRCA2 pathway can be functionally inactivated in sporadic breast cancer, suggesting that the pool of patients with defects in homologous recombinational repair is larger than initially expected. Citation Format: Simon N. Powell. Connections in the BRCA1-BRCA2 pathway of homologous recombination: Implications for breast cancer development and treatment [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY28-02
RAD52
BRCA2 Protein
Non-homologous end joining
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Summary Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single–molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP–dependent anti–recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55– Rad57 does not physically block the movement of Srs2. Instead, Rad55–Rad57 promotes rapid re– assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA–bound states, the rate of which is dynamically controlled though the opposing actions of Rad55–Rad57 and Srs2.
Chaperone (clinical)
FLP-FRT recombination
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