STAR Protocols
·
2023
·
IF 0
Zenan Zhou,Han Yang,Xinxin Liang,Tao Zhou,Qixiang Liu,Jiadong Wang,Weibin Wang
Summary C1orf112/FIRRM is a recently identified DNA damage repair factor that regulates RAD51 in homologous recombination through interacting with the anti-recombinase FIGNL1. Here, we describe steps for purifying C1orf112/FIRRM, FIGNL1, miBRCA2, and RAD51 proteins from Escherichia coli or Saccharomyces cerevisiae cells. We then detail procedures for reconstituting the disassembly of RAD51 filament by C1orf112/FIRRM-FIGNL1 in vitro and the antagonistic effect between C1orf112/FIRRM-FIGNL1 and miBRCA2 on RAD51 filament stabilization. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2023). 1
研究领域:
分子生物学
·
细胞生物学
·
遗传学
·
生物化学
✓ 已验证
DOI
PubMed
Translational Oncology
·
2025
·
IF 4.1
Zhe Qin,Fangzhou Ye,Jiayi Wang,Jun Jiang,Xiaohong Zhang,Huanqing Li,Li Feng
Background Cisplatin resistance significantly impedes the treatment of gastric cancer (GC). This work examined the possible therapeutic target status and function of BUB1B in controlling cisplatin resistance. Methods Following the identification of differentially expressed genes (DEGs), protein-protein interaction (PPI) network analysis was conducted using datasets from the Cancer Genome Atlas-stomach adenocarcinoma (TCGA-STAD), GSE51575, and GSE79973. Functional tests assessed the effect of BUB1B overexpression and knockdown on the GC cells. Enrichment analysis and RNA-seq identified pathways linked to BUB1B . Additionally, the function of BUB1B in GC cells resistant to cisplatin in regulating DNA repair was examined, as its relationship with Rad51 inhibitor (B02) in regulating cell cycle, proliferation, and apoptosis. The combined effects of Rad51 suppression and BUB1B overexpression on tumor development in cisplatin-resistant GC cells were further validated in vivo xenograft models. Results Significant overexpression of six critical overlapping genes was seen in GC tissues. The GC cell invasion, migration, and proliferation processes were improved by BUB1B overexpression, whereas BUB1B knockdown prevented these outcomes. Genes involved in DNA repair were downregulated by BUB1B knockdown, according to an RNA-seq study. BUB1B overexpression boosted cell survival via modulating cell cycle proteins, but BUB1B knockdown hampered DNA repair and increased death in cisplatin-resistant GC cells. Overexpression of BUB1B enhanced tumor development in vivo and counteracted the inhibitory effects of B02 on cell growth. Conclusion BUB1B enhances cisplatin resistance in gastric cancer by regulating DNA repair and cell cycle progression, suggesting that targeting BUB1B may be a feasible therapeutic strategy.
研究领域:
肿瘤学
·
分子生物学
·
癌症治疗
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF MOLECULAR MODELING
·
2015
·Vol. 21
·期 11
·1-7
·
IF 1.736
Zhao Dongxin,Lu Kui
Breast cancer susceptibility gene 2 (BRCA2)—a human tumor suppressor gene—is related to various malignancies such as breast and ovarian cancer. This gene can induce the key protein RAD51 recombinase, which is involved in homologous recombination with single-stranded DNA in the human body and can regulate RAD51 to complete the repair of damaged double-stranded DNA. Eight highly conserved BRC repeat motifs in BRCA2 protein serve as sites for the interaction between BRCA2 and RAD51. BRCA2 regulates RAD51 through these motifs. However, the mechanism of this interaction still requires further research. In this study, the BRC4 motif that demonstrated strong interaction with RAD51 was selected as template peptide. On the basis of known data regarding the crystal structure of the BRC4-RAD51(191–220) complex, a series of BRC4 mutants was designed using PyMOL software based on the sequence of BRC4, and polypeptides were synthesized by the Fmoc solid-phase method. After purification by reversed-phase high-performance liquid chromatography, the purity of the polypeptides reached >95 %. The primary determination of circular dichroism spectra showed that the polypeptides exhibited slight changes in secondary structure, which indicated that mutation on the non-conserved sites in BRC4 probably affected the interaction with BRC4. These findings will facilitate research on the interaction between targeting peptides and BRC4 mutants, as well the basic rules covering this interaction.
研究领域:
分子生物学
·
结构生物学
·
肿瘤学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CHEMICAL RESEARCH IN TOXICOLOGY
·
2016
·
IF 3.025
Xiufang Song,Qiong Shi,Zixuan Liu,Yawen Wang,Yuxin Wang,Erqun Song,Yang Song
We previously demonstrated that halogenated quinone induces DNA double strand breaks (DSBs) in a ROS-dependent manner, which coordinates with downstream repair cascade including nonhomologous end joining, base excision repair, and nucleotide excision repair. However, these error-prone processes may cause the potential risk of genome instability, and current has no information on how faithful repair route, such as homologous recombination (HR), was affected. RAD51 is a key protein in the HR pathway of DSBs repair. Here, we found that tetrachlorobenzoquinone (TCBQ) causes a time-dependent reverse U-shape biphasic trend of RAD51 expression. An increase in the early stage and a following decrease of RAD51 expression were found in both 12.5 and 25 μM TCBQ groups, wherein higher concentration faced a faster response. The upregulated RAD51 in the early phase suggested the attempting to repair TCBQ-induced DNA damage; however, the downregulation of RAD51 in the late phase implicated that the rescue probably be abandoned with severe DNA damage. This phenomenon is a general toxic manner of TCBQ regardless of cell type. Surprisingly, TCBQ showed minimum effect on RAD51 mRNA (or protein) synthesis as well as RAD51 degradation. Specific inhibition of RAD51 by siRNA amplified TCBQ-induced DNA damage and cytotoxicity, while cells with enhanced RAD51 expression resisted TCBQ-induced toxicity. The modulation of RAD51 is correlated with p53 level, which suggests p53 has a role in TCBQ-induced RAD51 clearance. Together, our data suggested that TCBQ increases genome instability and cell death through a unique mechanism of inducing DNA damage and inhibiting DNA repair.
研究领域:
毒理学
·
分子生物学
·
遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
PHARMACEUTICAL CHEMISTRY JOURNAL
·
2024
·
IF 0.8
Pan Boyuan,Fu Linna,Du Heng,Liu Guangbin,Duan Bingchao,Lu Kui
RAD51 is a core factor for homologous recombination to repair DNA double-strand breaks and overexpressed in breast cancer cells. Truncated peptide BRC4 (1523-1537) was obtained by computer simulation which had the highest binding free energy targeting RAD51. To enhance the binding affinity to the target protein, six nicotinic acid derivatives were modified at the N -terminal of BRC4 (1523-1537) by Fmoc solid-state synthesis to obtain nicotinamide-modified peptides. The interaction of RAD51 (181-200) with BRC4 (1523-1537) and nicotinamide-modified peptides was verified by circular dichroism (CD) spectroscopy and fluorescence spectroscopy. In conclusion, modifying small molecule pharmacophores can improve binding ability. According to spectral results, 2-chloro-5-fluoronicotinic acid modified BRC4 (1523-1537) has the most significant influence on the secondary structure of RAD51 (181-200); binding constant is 1.1×10 4 L·mol -1 . Cell experiments showed that BRC4 (1523-1537) modified with nicotinic acid N-oxide had the best inhibitory effect on the proliferation of MDA-MB-231 cells.
研究领域:
生物化学
·
癌症研究
·
药物设计
✓ 已验证
产品关联
DOI
高影响因子
International Journal of Peptide Research and Therapeutics
·
2021
·Vol. 27
·期 2
·1343-1350
·
IF 1.931
Zhao Yi,Sun Guangming,Yuan Libo,Li Jinlei,Lu Kui
Breast cancer susceptibility gene 1 (BRCA1) is an important tumor suppressor gene in the human which associated with a variety of tumor suppressor genes to inhibit the growth of malignant tumors. Breast cancer is closely related to BRCA1 mutations in humans. Four BRCA1 (856-871) mutants were screened that can interact well with RAD51 (158-180) using computer to simulate mutations at Y 856, R 866 and Q 867. These mutants were synthesized by using Fmoc solid-phase synthesis, and purified by liquid chromatography. According to the results of fluorescence spectroscopy, R866W has the strongest binding ability for the above peptides and RAD51 (158-180). Based on the circular dichroism spectrum results, the sequence of the interaction ability of the above peptides on the secondary structure of RAD51 (158-180) is Y856P, Q867C > Y856P, R866W ≈ R866W > Y856P > BRCA1. All the spectral results show that R866W have the strongest interaction ability with RAD51(158-180).
研究领域:
分子生物学
·
肿瘤学
·
生物化学
✓ 已验证
产品关联
DOI
高影响因子
International Journal of Peptide Research and Therapeutics
·
2019
·Vol. 26
·期 1
·121-128
·
IF 1.219
Wang Mengwei,Lv Mingxiu,Lu Kui,Liu Guangbin,Mai Wenpeng,Yu Bo,Lou Yudan
Six breast cancer susceptibility gene 1 (BRCA1) peptide fragments were designed by removing N-terminal amino acid residues of BRCA1(846–871). Peptide fragments BRCA1(846–871), BRCA1(852–871), BRCA1(854–871), BRCA1(856–871), BRCA1(857–871) and BRCA1(861–871) were obtained by Fmoc solid-phase synthesis, purified by reversed-phase high-performance liquid chromatography and characterized by electrospray ionization mass spectrometry. The interaction between BRCA1 fragment peptides and RAD51(181–200) target peptides were investigated using circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and microscale thermophoresis (MST). The results of CD spectroscopy showed that the secondary structures of RAD51(181–200) were altered after the addition of BRCA1 peptide fragments. The fluorescence results proved that all six BRCA1 peptide fragments BRCA1(846–871), BRCA1(852–871), BRCA1(854–871), BRCA1(856–871), BRCA1(857–871) and BRCA1(861–871) could bind to RAD51(181–200) independently, their quenching processes were static, and the binding constant of peptide fragments with RAD51(181–200) were 4.01 × 10 5 L mol −1 , 7.22 × 10 5 L mol −1 , 7.84 × 10 5 L mol −1 , 1.1 × 10 4 L mol −1 , 6.19 × 10 3 L mol −1 , 6.18 × 10 3 L mol −1 . The results of MST displayed that BRCA1(854–871) could combine with RAD51(181–200), and the dissociation constant K d was 126.44 µM. Compared with other five peptide fragments, the interaction between BRCA1(854–871) and RAD51(181–200) was much stronger.
研究领域:
生物化学
·
分子生物学
·
癌症研究
✓ 已验证
DOI
高影响因子
AMINO ACIDS
·
2020
·Vol. 52
·期 5
·831-839
·
IF 3.063
Zhao Dongxin,Lu Kui,Liu Guangbin,Zhu Hanjing,Ma Li,Qi Jianzhao,Yuan Libo
BRCA2 is an important tumor suppressor gene that plays a critical role in preserving the stability of cellular genetic information, participating in DNA repair by engaging in binding interactions with RAD51 proteins. However, the lack of structural data on BRCA2 and RAD51 makes the study of their interaction mechanism still a great challenge. We characterize the structure of the BRC8–RAD51 complex using ZDOCK protein docking software and identify the potential non-conserved active site of BRC8 via virtual alanine scanning, utilizing the obtained results to synthesize BRC8, its six analogous peptides (BRC8-1 to BRC8-6), and critical peptide fragment of RAD51 (RAD51(231–260)) by Fmoc solid-phase synthesis. The analogous peptides are found to exhibit a secondary structure significantly different from that of BRC8 by circular dichroism spectroscopy, which indicates that mutation sites determined by computer-aided simulation correspond to key amino acid residues substantially affecting polypeptide structure. On the other hand, the secondary structure of RAD51(231–260) was also considerably influenced by its interaction with BRC8 and analogs, e.g., the fraction of the α-helical structure in RAD51(231–260) increased to 23.6, 15.1, and 13.5% upon interaction with BRC8-1, BRC8-3, and BRC8-6, respectively. The results show that the properties of C-terminal amino acid residues significantly influence peptide–peptide interactions, in agreement with the results of virtual alanine scanning. Therefore, computer-aided simulation was confirmed to be a technique that is useful for narrowing down the range of sites responsible for interactions between peptides or proteins, and provides new inspirations for the design of peptides with strong interactions.
研究领域:
生物化学
·
分子生物学
·
结构生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
International Journal of Peptide Research and Therapeutics
·
2019
·Vol. 26
·期 4
·1991-1999
·
IF 1.219
Cao Mengjie,Lu Kui,Liu Guangbin,Zhao Dongxin,Qi Jianzhao
Affecting the repair function of RAD51 can effectively reverse tumor resistance to chemotherapy and radiotherapy. There is interaction between RAD51 181−200 and BRCA1 846−871 . Using the computer to simulate single mutation of lysine at 862 position of BRCA1 846−871 , six BRCA1 846−871 mutants with significant interaction with RAD51 181−200 were screened out. These mutant peptides were obtained by 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) method. All peptides were purified and characterized by liquid chromatography and ESI-MS. In this study, the interaction between RAD51 181−200 and BRCA1 846−871 or mutant peptides was investigated using circular dichroism spectroscopy (CD) and fluorescence spectroscopy. The CD results shown that the secondary structure change of RAD51 181−200 induced by BRCA1 846−871 , BRCA1 846−871 K862F ( F ), BRCA1 846−871 K862G ( G ), BRCA1 846−871 K862E ( E ) and BRCA1 846−871 K862N ( N ) was obvious, which the α-helix content of RAD51 181−200 decreased from 58.3–36.1%, 58.3–36.2%, 58.3–36.4%, 58.3–36.5% and 58.3–36.0%, respectively. While, the addition of BRCA1 846−871 K862P ( P ) and BRCA1 846−871 K862Y ( Y ) hardly changed the α-helix content of RAD51 181−200 . From the fluorescence results, the binding constants of F and Y are 1.00 × 10 5 M −1 and 1.16 × 10 5 M −1 respectively, which were both larger than that of BRCA1 846−871 (9.01 × 10 2 M −1 ). The binding constant of RAD51 181−200 with E (7.45 × 10 3 M −1 ) is slightly higher than that of BRCA1 846−871 . The binding constants of RAD51 181−200 with P (1.24 × 10 3 M −1 ) and N (1.26 × 10 3 M −1 ) are similar to that of BRCA1 846−871 . However, the K b value of G (1.51 × 10 2 M −1 ) is less than that of BRCA1 846−871 . Comparing all spectral data, F is the most significant one interacting with RAD51 181−200 . These findings could provide direction for peptide processing and application.
研究领域:
生物化学
·
分子生物学
·
肿瘤学
✓ 已验证
产品关联
DOI
高影响因子
AMINO ACIDS
·
2019
·Vol. 52
·期 3
·361-369
·
IF 2.52
Li Jinlei,Lu Kui,Lv Mingxiu,Liu Guangbin,Ma Li,Qi Jianzhao
Breast cancer susceptibility gene 2 ( BRCA2 ) is an important tumor suppressor, which is participated in repair of damaged DNA by its highly conserved BRC repeat motifs regulating RAD51 protein homologous recombination and thereby preventing cell carcinogenesis. In this study, the BRCA2(1524–1548)–RAD51(241–260) complex structure was obtained based on PDB bank data 1N0W, which provided the basis for site-specific mutation of BRCA2(1524–1548). The BRC4 and BRC4 analogous peptides were synthesized, and the interaction between BRC peptide and RAD51(241–260) was studied by fluorescence spectroscopy, circular dichroism spectroscopy and microscale thermophoresis (MST). The results of circular dichroism showed that the changes in secondary structures of RAD51(241–260) occurred after adding BRC4 analogous peptides, and the α-helix content increased significantly. Fluorescence spectral data demonstrated that the model of BRC peptide binding to RAD51(241–260) was static quenching, and the binding constants of BRC4, P1, P2, P4 with RAD51(241–260) were 1.647 × 10 −4 L mol −1 , 2.532 × 10 −4 L mol −1 , 3.161 × 10 −4 L mol −1 , 1.705 × 10 −4 L mol −1 , respectively. The results of MST indicated that P2 and RAD51(241–260) have better affinity for dissociation constant 44.286 μM. The strongest affinity between P2 and RAD51(241–260) indicated that the mutation of amino acid residue constituting BRC α-helix affects the structure and interaction of BRC peptide and RAD51(241–260).
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Acta Pharmaceutica Sinica B
·
2025
·
IF 14.6
Wei Xu,Minmin Shen,Junjie Ma,Chuanlin Peng,Shanshan Wu,Xinxin Yang,Jiahe Wu,Youyou Yan,Nengming Lin,Jianqing Gao,Bo Zhang
Dysregulation of cytoskeletal proteins has been found in response to DNA damage stress, yet the functional role of cytoskeletal proteins in DNA repair remained unexplored. Here, we found that DNA-damaging agents induced substantial upregulation of smooth muscle-specific cytoskeletal protein smoothelin (SMTN) in colorectal cancer (CRC) cells. Silencing SMTN abrogated G2/M arrest, exacerbated DNA damage, and markedly enhanced the chemosensitivity of CRC cells to various DNA-damaging agents. Notably, SMTN could rapidly accumulate at DNA damage sites within 1 min after laser irradiation, which was indispensable for the initiation of homologous recombination (HR) repair. Mechanistically, SMTN stabilized RAD51 by disrupting its interaction with its E3 ubiquitin ligase HUWE1, thereby maintaining the process of HR repair. To explore the therapeutic role of SMTN, customized cell membrane infused biomimetic liposomes were constructed to ensure rapid delivery of SMTN siRNA specifically into HCT-116 cells, yielding significantly enhanced anti-cancer effects of irinotecan and fuzuloparib both in vitro and in vivo . To summarize, our findings revealed a novel function of SMTN in DNA damage repair and provided a therapeutic strategy of targeting SMTN to enhance the efficacy of DNA damage agents.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOMATERIALS
·
2021
·
IF 12.479
Qi Xu,Hao Zhang,Hanghang Liu,Yaobao Han,Weibao Qiu,Zhen Li
Radio-resistance of glioblastoma (GBM) remains a leading cause of radiotherapy failure because of the protective autophagy induced by X-Ray irradiation and tumor cells’ strong capability of repairing damaged DNA . It is of great importance to overcome the radio-resistance for improving the efficacy of radiotherapy. Herein, we report the novel mechanism of core-shell copper selenide coated gold nanoparticles ( [email protected] 2- x Se NPs) inhibiting the protective autophagy and DNA repair of tumor cells to drastically boost the radiotherapy efficacy of glioblastoma. We reveal that the core-shell [email protected] 2- x Se NPs can inhibit the autophagy flux by effectively alkalizing lysosomes . They can increase the SQSTM1/p62 protein levels of tumor cells without influencing their mRNA. We also reveal that [email protected] 2- x Se NPs can increase the ubiquitination of DNA repair protein Rad51, and promote the degradation of Rad51 by proteasomes to prevent the DNA repair. The simultaneous inhibition of protective autophagy and DNA repair significantly suppress the growth of orthotopic GBM by using radiotherapy and our novel [email protected] 2- x Se NPs. Our work provides a new insight and paradigm to significantly improve the efficacy of radiotherapy by rationally designing theranostic nano-agents to simultaneously inhibit protective autophagy and DNA repair of tumor cells.
研究领域:
肿瘤学
·
纳米技术
·
放射生物学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）