生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (4): 34-.doi: 10. 3969 / j. issn. 2095-1736. 2021. 04. 034

• 研究报告 • 上一篇    下一篇

分枝结构特异性内切酶-1(FEN1)突变体N266D表达纯化及功能分析

  

  1. 1.苏州卫生职业技术学院苏州市检验医学生物技术重点实验室,苏州215009; 2.南京师范大学生命科学学院江苏省分子与医学生物技术重点实验室,南京210023
  • 出版日期:2021-08-18 发布日期:2021-08-18
  • 通讯作者: 刘柏松,博士,副教授,主要研究方向为肿瘤学,E-mail:liusongbai@126.com; 郭志刚,教授,博士,主要研究方向为肿瘤学,E-mail:guozgang@gmail.com
  • 作者简介:邱秀芹,硕士,副教授,研究方向为分子生物学,E-mail:qxq199609@163.com
  • 基金资助:
    国家自然科学基金项目(31701198);江苏省自然科学基金项目(BK20170386);

Expression,purification and function alanalysis of Flapendonuclease-1(N266D)mutant

  1. 1. Suzhou Key Laboratory of Medical Biotechnology, Suzhou Vocational Health College, Suzhou 215009, China;2. Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Science,Nanjing Normal University, Nanjing 210023, China
  • Online:2021-08-18 Published:2021-08-18

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摘要： 人源分枝结构特异性内切酶-1(Flapendonuclease1,FEN1)作为DNA复制及DNA修复过程中重要的参与者摘,在维护基因组稳定和完整性方面具有不可替代的作用。尽管FEN1在很多肿瘤细胞中的表达都发生异常,但FEN1基因体细胞突变或遗传突变事件在人体细胞中却很少发生,发表的文献报道较少。前期在白血病患者骨髓细胞中发现了FEN1基因的一种N266D杂合突变体,基于此,成功构建了FEN1-N266D在原核及真核中的表达重组质粒,纯化了原核FEN1-WT、FEN1-N266D蛋白;通过TAMRA标记的DNA底物,检测了FEN、GEN和EXO的3种酶活性,结果发现N266D突变体的FEN、GEN及EXO活性基本不变;RTCA方法检测发现N266D蛋白过表达使细胞增殖能力受到抑制。通过以上对FEN1突变体N266D蛋白的核酸酶活性及对细胞增殖能力影响的分析,为进一步探讨FEN1突变体N266D在肿瘤发生进展中的潜在功能奠定了基础

关键词: FEN1, 基因突变体, 核酸酶活性, 细胞增殖

Abstract: As a central component during DNA replication and DNA repair process, humanflapendonuclease-1(FEN1) plays indis-pensable roles in genome stability and integrity. AlthoughFEN1expression level is dysregulated in many types of tumor cells, mutation events ofFEN1gene are rare to be reported in the study paper. We previously detected one N266D heterozygotic mutant in the hemato-poietic cells of a leukemia patient. In this study, we successfully constructed N266D expressed plasmids, and purified FEN1-WT and N266D proteins. We also detected the nuclease activities of N266D and FEN1-WT by TAMRA tagged DNA substrates and determinedthat there was no obvious difference between them. RTCA method determined that the proliferation capacity of 293T cells was sup-pressed when overexpressed with N266D mutant. Based on our results of nuclease activities and cell proliferation capacity of N266Dmutant, it is possible to dig the potential roles of N266D mutant during tumor development in future.

Key words: FEN1, mutant, nuclease activity, cell proliferation

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