STAR Protocols
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2023
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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
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细胞生物学
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遗传学
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生物化学
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DOI
PubMed
JOURNAL OF MOLECULAR MODELING
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2015
·Vol. 21
·期 11
·1-7
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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
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结构生物学
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肿瘤学
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产品关联
DOI
PubMed
高影响因子
FEBS LETTERS
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2018
·Vol. 592
·期 20
·3438-3445
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IF 2.999
Dongxin Zhao,Kui Lu,Guangbin Liu,Li Ma,Hanjing Zhu,Juan He
Mutations in breast cancer susceptibility gene 2 ( BRCA2 ) can lead to chromosomal instability and result in breast cancer, which is strongly associated with p53 mutations. Here, based on the crystal structure of BRC4 and p53, the spatial structure of BRC2 and p53 (171–192) was simulated, providing structural basis for the site-specific mutation of BRC2. The BRC analogous peptides and p53 (171–192) were synthesized, and the interaction between the mutant peptide and p53 (171–192) was studied using circular diachronic spectroscopy and fluorescence spectroscopy. The results show that the mutations of amino acid residues constituting the BRC2 α-helix significantly affect the structure and interaction of BRC analogs and p53 (171–192), which provides support for understanding the structure of the BRC repeat motifs and its interaction pattern with p53.
研究领域:
分子生物学
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癌症研究
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生物化学
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产品关联
DOI
PubMed
高影响因子
AMINO ACIDS
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2020
·Vol. 52
·期 5
·831-839
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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
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分子生物学
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结构生物学
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产品关联
DOI
PubMed
高影响因子
FREE RADICAL BIOLOGY AND MEDICINE
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2024
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IF 7.1
Yixing Huang,Xiao Li,Shengfeng Xu,Dan Zu,Haidong Liu,Hanyi He,Qimei Bao,Yanhua He,Chen Liang,Yin Shi,Xiangdong Cheng,Yaoshu Teng,Zu Ye
Nanoplastics (NPs), which are characterized by plastic particles smaller than one micrometer, have emerged as pervasive environmental pollutants, raising concerns about their potential toxicity to living organisms. Numerous investigations have highlighted the tendency of NPs to accumulate in organs, resulting in toxic effects. Despite polyvinyl chloride (PVC) being one of the most prevalent NPs, its impact on the esophagus and the associated underlying mechanisms remain largely unknown. In this study, we investigated the impact of PVC NPs on the esophagus and found that PVC NPs exposure induces oxidative stress and elicits DNA damage responses. Further analysis revealed that PVC NPs inhibit the homology-directed repair (HDR) pathway by suppressing the expression of breast cancer susceptibility gene 2 ( BRCA2 ) and growth factor receptor-bound protein 2 ( GRB2 ), resulting in genomic instability. Additionally, the release of free DNA activates cGAS-STING and the downstream NF-κB signaling, elevating inflammatory factors and chemokines, which further leads to cellular senescence. In vivo experiments corroborated these findings, showing that PVC NPs induced oxidative stress, inflammation, and cellular senescence, subsequently impacting mouse behavior. This study contributes novel insights into the health risks associated with PVC NPs exposure and identifies potential therapeutic targets.
研究领域:
毒理学
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分子生物学
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细胞生物学
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环境科学
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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）