泛素连接酶Brl2在DNA双链断裂修复中的作用分析

doi:10.16288/j.yczz.22-031

遗传 ›› 2022, Vol. 44 ›› Issue (7): 609-617.doi: 10.16288/j.yczz.22-031

泛素连接酶Brl2在DNA双链断裂修复中的作用分析

刘晓琴¹(), 常斐然², 刘思杰², 伍斐², 孔道春²

1. 山西大同大学脑科学研究所,大同 037009
2. 北京大学生命科学学院,北京 100871

收稿日期:2022-02-14 修回日期:2022-04-25 出版日期:2022-07-20 发布日期:2022-05-19
通讯作者: 刘晓琴 E-mail:xiaoqinliu@sxdtdx.edu.cn
基金资助:
山西省高等学校科技创新项目编号(2020L0497);山西大同大学博士科研启动经费资助编号(2019-B-20)

The functional analysis of the ubiquitin ligase Brl2 in the repair of DNA double-strand breaks

Xiaoqin Liu¹(), Feiran Chang², Sijie Liu², Fei Wu², Daochun Kong²

1. Institute of Brain Science, Shanxi Datong University, Datong 037009, China
2. College of Life Sciences, Peking University, Beijing 100871, China

Received:2022-02-14 Revised:2022-04-25 Online:2022-07-20 Published:2022-05-19
Contact: Liu Xiaoqin E-mail:xiaoqinliu@sxdtdx.edu.cn
Supported by:
Supported by the Scientific and Technological Innovation Program of Higher Education Institutions in Shanxi No(2020L0497);Doctoral Research Start-up Funds of Shanxi Datong University No(2019-B-20)

摘要/Abstract

摘要：

组蛋白H2B单泛素化在基因转录、DNA复制及损伤修复中发挥着重要的调控作用。在裂殖酵母(Schizosaccharomyces pombe)中,Brl2作为一个泛素化连接酶,调节H2B的119位赖氨酸的单泛素化。目前,有关Brl2在DNA损伤修复中的作用研究较少,本研究利用药物喜树碱(camptothecin, CPT)处理裂殖酵母产生高毒性的DNA双链断裂(DNA double-strand breaks, DSBs),探索Brl2在DSB修复过程中的作用。研究发现,brl2基因缺失的菌株对CPT高度敏感,并导致细胞内DNA自发重组频率下降。荧光分析表明Brl2和重组修复蛋白Rad52共定位到DSB处,且Brl2促进Rad52在DSB处的募集。在CPT产生的DSB条件下,Brl2会发生磷酸化。以上研究发现揭示了Brl2在DSB修复过程中起重要作用,为具体阐明Brl2在DNA同源重组及双链断裂修复的分子机制奠定了进一步研究的基础。

关键词: 组蛋白H2B, Brl2, DNA双链断裂修复, 喜树碱, 裂殖酵母

Abstract:

Mono-ubiquitination of histone H2B plays a critical role in the regulation of gene transcription, DNA replication, and DNA damage repair. In Schizosaccharomyces pombe, Brl2 is an E3 ubiquitin ligase and required for the ubiquitination of H2B at lysine residue 119. Currently, there are few studies related to the function of Brl2 in DNA damage repair. Using camptothecin (CPT) to induce DNA double-strand breaks (DSBs) in S. pombe, we investigated the effect of Brl2 on DSB repair, and found that brl2-null mutants showed greater sensitivity to CPT when compared with wild-type (WT) cells, as well as having a drastically reduced spontaneous recombinant frequency. The fluorescent analysis demonstrated that Brl2 was co-localized with the recombination factor Rad52 at DSBs. Moreover, Brl2 promoted the recruitment of Rad52 to DSBs. Under CPT-induced DSBs, Brl2 was phosphorylated. These findings indicate that Brl2 plays a critical role in DNA homologous recombination and its mediated repair of DSBs.

Key words: histone H2B, Brl2, DSB repair, CPT, S. pombe

刘晓琴, 常斐然, 刘思杰, 伍斐, 孔道春. 泛素连接酶Brl2在DNA双链断裂修复中的作用分析[J]. 遗传, 2022, 44(7): 609-617.

Xiaoqin Liu, Feiran Chang, Sijie Liu, Fei Wu, Daochun Kong. The functional analysis of the ubiquitin ligase Brl2 in the repair of DNA double-strand breaks[J]. Hereditas(Beijing), 2022, 44(7): 609-617.

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菌株	交配型	基因型
LD330	h^-	Ura4-D18
rad3Δ	h^-	leu-32, ura4-D18, ade6-M210, his-D1, rad3::leu2
cds1Δ	h⁺	ade6-704, cds1::kanMX6, leu1-32, ura4-D18
brl2Δ	h^-	brl2::ura4, Ura4-D18
brl2Δ rad3Δ	h^-	brl2::ura4, leu-32, ura4-D18, ade6-M210, his-D1, rad3::leu2
brl2Δ cds1Δ	h⁺	brl2::ura4, ade6-704, cds1::kanMX6, leu1-32, ura4-D18
YKM619	h^-	ade6-M375 int:pUC8/ura4⁺/ade6-469 ura4-D18
brl2Δ-YKM619	h^-	brl2::kanMX6, ade6-M375 int: pUC8/ura4⁺/ade6-469 ura4-D18
rad3Δ-YKM619	h^-	rad3::hph, ade6-M375 int: pUC8/ura4⁺/ade6-469 ura4-D18
rad3Δ brl2Δ-YKM619	h^-	brl2::kanMX6, Rad3::hph, ade6-M375 int: pUC8/ura4⁺/ade6-469 ura4-D18
FY18445	h^-	rad52:EYFP:kanr leu1-32 ura4-D18
brl2Δ- FY18445	h^-	brl2::ura4, rad52:EYFP:kanr leu1-32 ura4-D18
DY1012	h^-	Leu-32 his3-D1 ura4-294 arg3Δ::HOsite-natMX ars::P41nmt-HO(his3+)lys-131::Pdis1-mCherry- LacI(lys1+)erg7ter::lacOrepeat(ura4+)rad52-2XCFP::hphMX
brl2-3HA	h^-	brl2::brl2-3HA-ura4, Ura4-D18

泛素连接酶Brl2在DNA双链断裂修复中的作用分析

The functional analysis of the ubiquitin ligase Brl2 in the repair of DNA double-strand breaks

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