|本期目录/Table of Contents|

[1]龚晓昕,沈苗苗,李徐梓超,等.人源解旋酶样转录因子的表达、纯化和功能研究[J].天津医科大学学报,2022,28(01):35-39.
　GONG Xiao-xin,SHEN Miao-miao,LI Xu-zi-chao,et al.Expression，purification of human helicase-like transcription factor[J].Journal of Tianjin Medical University,2022,28(01):35-39.
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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
28卷
期数:
2022年01期
页码:
35-39
栏目:
基础研究
出版日期:
2022-01-20

文章信息/Info

Title:
Expression，purification of human helicase-like transcription factor
文章编号:
1006-8147（2022）01-0035-05
作者:
龚晓昕; 沈苗苗; 李徐梓超; 向嵩
（天津医科大学基础医学院生物化学与分子生物学系，天津 300070）
Author(s):
GONG Xiao-xin; SHEN Miao-miao; LI Xu-zi-chao; XIANG Song
（Department of Biochemistry and Molecular Biology，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China）
关键词:
解旋酶样转录因子; 蛋白质表达; 蛋白质纯化; 泛素连接酶活性; DNA复制压力应对
Keywords:
helicase-like transcription factor; protein expression; protein purification; ubiquitin ligase; DNA replication stress tolerance
分类号:
Q51
DOI:
-
文献标志码:
A
摘要:
目的：解旋酶样转录因子（HLTF）在人体细胞的DNA复制压力应对中发挥重要作用，对其结构和功能的研究需要大量、高纯度且折叠正确的蛋白质。方法：通过优化HLTF基因的密码子偏好，构建HLTF蛋白的大肠杆菌表达系统；通过亲和层析、离子交换层析、凝胶过滤层析等方法纯化HLTF；采用体外实验研究HLTF的生化活性。结果：成功构建HLTF蛋白的大肠杆菌表达系统，通过一个4步的纯化，获得高纯度的HLTF蛋白质，体外功能实验表明大肠杆菌表达的HLTF具有完整的生化活性，提示蛋白折叠正确。本研究发现HLTF的泛素连接酶活性对底物与泛素的连接方式有较大的容忍度。结论：建立了一种高效制备大量、高纯度且折叠正确的HLTF的方法，为深入理解其泛素连接酶活性提供线索，为其结构和功能研究奠定基础。
Abstract:
Objective：The helicase-like transcription factor（HLTF） plays critical roles in human cells′ response to DNA replication stresses；structural and functional studies of this important protein require significant amounts of pure and correctly folded HLTF. Methods: This study optimized the codon preference in the HLTF gene to construct an Escherichia coli-based HLTF expression system. Recombinant HLTF was purified by affinity chromatography，ion-exchange chromatography and gel filtration chromatography. In vitro experiments were carried out to probe the biochemical activities of the recombinant HLTF. Results: This Escherichia coli-based HLTF expression system was successfully constructed，and the high-purity HLTF protein was obtained by a 4-step purification procedure. In vitro functional experiments showed that the HLTF expressed by Escherichia. coli had complete biochemical activity，indicating that it is correctly folded. It was found that ubiquitin ligase activity of HLTF was insensitive towards the ubiquitin-substrate linkage. Conclusion: Here presents an efficient protocol that produces large quantities of pure and correctly folded HLTF，as well as insights into HLTF′s ubiquitin ligase activity.This study provides a starting point for future structural and functional studies of this important protein.

参考文献/References:

[1] ZEMAN M K，CIMPRICH K A. Causes and consequences of replication stress [J]. Nat Cell biol，2014，16（1）：2-9.
[2] MARIANS K J. Lesion bypass and the reactivation of stalled replication forks [J]. Annu Rev Biochem，2018，87:217-238.
[3] CORTEZ D. Replication-coupled DNA repair[J]. Mol Cell，2019， 74（5）：866-876.
[4] PASERO P，VINDIGNI A. Nucleases acting at stalled forks：how to reboot the replication program with a few shortcuts[J]. Annu Rev Genet，2017，51:477-499.
[5] BRANZEI D，PSAKHYE I. DNA damage tolerance[J]. Curr Opin Cell Biol，2016，40:137-144.
[6] CHATTERJEE N，WALKER G C. Mechanisms of DNA damage，repair，and mutagenesis [J]. Environ Mol Mutagen，2017，58（5）：235-263.
[7] SALE J E. Translesion DNA synthesis and mutagenesis in eukaryotes[J]. Cold Spring Harb Perspect Biol，2013，5（3）：a012708.
[8] UNK I，HAJD■ I，F?魣TYOL K，et al. Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination[J]. Proc Natl Acad Sci U S A，2008，105（10）：3768-3773.
[9] LEE K Y，MYUNG K. PCNA modifications for regulation of post-replication repair pathways [J]. Mol Cells，2008，26（1）：5-11.
[10] MOTEGI A，LIAW H J，LEE K Y，et al. Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks [J]. Proc Natl Acad Sci U S A，2008，105（34）：12411-12416.
[11] BLASTY?魣K A，HAJD■ I，UNK I，et al. Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA [J]. Mol Cell Biol，2010，30（3）：684-693.
[12] NEELSEN K J，LOPES M. Replication fork reversal in eukaryotes：from dead end to dynamic response[J]. Nat Rev Mol Cell Biol，2015， 16（4）：207-220.
[13] UNK I，HAJD■ I，BLASTY?魣K A，et al. Role of yeast Rad5 and its human orthologs，HLTF and SHPRH in DNA damage tolerance [J]. DNA Repair （Amst），2010，9（3）：257-267.
[14] ELSERAFY M，ABUGABLE A A，ATTEYA R，et al. Rad5，HLTF，and SHPRH：a fresh view of an old story[J]. Trends Genet，2018， 34（8）：574-577.
[15] ACHAR Y J，BALOGH D，NECULAI D，et al. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling [J]. Nucleic Acids Res，2015，43（21）：10277-10291.
[16] HISHIKI A，HARA K，IKEGAYA Y，et al. Structure of a novel DNA-binding domain of helicase-like transcription factor（HLTF） and its functional implication in DNA damage tolerance[J]. J Biol Chem，2015，290（21）：13215-13223.
[17] KILE A C，CHAVEZ D A，BACAL J，et al. HLTF′s ancient HIRAN domain binds 3′ DNA ends to drive replication fork reversal[J]. Mol Cell，2015，58（6）：1090-1100.
[18] SHEN M，DHINGRA N，WANG Q，et al. Structural basis for the multi-activity factor Rad5 in replication stress tolerance[J]. Nat Commun，2021，12（1）：321.
[19] KIM H J，KIM H J. Yeast as an expression system for producing virus-like particles：what factors do we need to consider?[J]. Lett Appl Microbiol，2017，64（2）：111-123.
[20] VILLALOBOS A，NESS J E，GUSTAFSSON C，et al. Gene designer：a synthetic biology tool for constructing artificial DNA segments[J]. BMC Bioinformatics，2006，7285.
[21] STENSTR?魻M C M，HOLMGREN E，ISAKSSON L A. Cooperative effects by the initiation codon and its flanking regions on translation initiation[J]. Gene，2001，273（2）：259-265.
[22] BARKER S C，KASSEL D B，WEIGL D，et al. Characterization of pp60c-src tyrosine kinase activities using a continuous assay：autoactivation of the enzyme is an intermolecular autophosphorylation process [J]. Biochemistry，1995，34（45）：14843-14851.
[23] LAURSEN B S，S?覫RENSEN H P，MORTENSEN K K，et al. Initiation of protein synthesis in bacteria[J]. Microbiol Mol Biol Rev，2005， 69（1）：101-123.
[24] TENAILLON O，SKURNIK D，PICARD B，et al. The population genetics of commensal Escherichia coli [J]. Nat Rev Microbiol，2010，8（3）：207-217.
[25] HARACSKA L，UNK I，PRAKASH L，et al. Ubiquitylation of yeast proliferating cell nuclear antigen and its implications for translesion DNA synthesis [J]. Proc Natl Acad Sci U S A，2006，103（17）：6477-6482.
[26] HOEGE C，PFANDER B，MOLDOVAN G L，et al. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO[J]. Nature，2002，419（6903）：135-141.

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备注/Memo

备注/Memo:
基金项目 国家自然科学基金（31870769，32071205）
作者简介 龚晓昕（1995-），女，硕士在读，研究方向：结构生物学；通信作者：向嵩，E-mail：xiangsong@tmu.edu.cn。
更新日期/Last Update: 2022-01-20