nature

当前位置: Nature » 分子生物学 » 正文

Sci Rep:上海交大由德林研究组发表DNA磷硫酰化修饰生物化学研究进展

摘要 : 7月27日,《scientific reports》杂志在线发表了上海交通大学生命科学技术学院、微生物代谢国家重点实验室由德林教授研究小组曹博(第一作者)的研究论文“In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex” 。

7月27日,《scientific reports》杂志在线发表了上海交通大学生命科学技术学院、微生物代谢国家重点实验室由德林教授研究小组曹博(第一作者)的研究论文“In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex” 。该研究首次在体外再现了DNA磷硫酰化修饰生物反应,并发现磷硫酰化修饰过程中形成了600 kDa的蛋白复合体用于底物识别和催化。

20150729-2

DNA磷硫酰化是硫原子取代DNA骨架非桥联氧原子的一种新型表观遗传学修饰,这种修饰广泛存在于原核生物中。由德林教授研究小组最近阐明其5个修饰基因中dptB基因为负调控基因,在转录水平上调控DNA磷硫酰化修饰基因的表达(Cheng et al, Molecular Microbiology 2015, doi: 10.1111/mmi.13096),但其它4个修饰基因在DNA磷硫酰化修饰过程中的具体功能以及修饰蛋白对DNA底物的识别机制目前尚不清楚。

研究通过构建沙门氏菌的全细胞裂解液催化体系,在体外再现了DNA磷硫酰化修饰反应。实验结果发现,GAAC/GTTC为修饰反应的保守序列,其两侧序列对修饰没有影响,双链DNA可以在体外被修饰,但单链DNA不能被修饰。研究还发现参与修饰反应的DptC、DptD和DptE 3个蛋白形成了稳定的600 kDa复合体,用于底物识别和催化。该研究首次展示了在体外DNA磷硫酰化修饰中酶是以蛋白复合物的形式实现修饰功能,为进一步深入理解DNA磷硫酰化的生物化学过程奠定了重要基础。

原文链接:

In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex

原文摘要:

A wide variety of prokaryotes possess DNA modifications consisting of sequence-specific phosphorothioates (PT) inserted by members of a five-gene cluster. Recent genome mapping studies revealed two unusual features of PT modifications: short consensus sequences and partial modification of a specific genomic site in a population of bacteria. To better understand the mechanism of target selecion of PT modifications that underlies these features, we characterized the substrate recognition of the PT-modifying enzymes termed DptC, D and E in a cell extract system from Salmonella. The results revealed that double-stranded oligodeoxynucleotides underwent de novo PT modification in vitro, with the same modification pattern as in vivo, i. e., GpsAAC/GpsTTC motif. Unexpectedly, in these in vitro analyses we observed no significant effect on PT modification by sequences flanking GAAC/GTTC motif, while PT also occurred in the GAAC/GTTC motif that could not be modified in vivo. Hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA. The PT-modifying enzymes were then found to function as a large protein complex, with all of three subunits in tetrameric conformations. This study provided the first demonstration of in vitro DNA PT modification by PT-modifying enzymes that function as a large protein complex.

来源: Scientific Reports 浏览次数:0

热门文章TOP

RSS订阅 - 填写您的邮件地址,订阅我们的精彩内容: - 网站地图
网站联系电话:020-87540820 备案号:粤ICP备11050685号-8 增值电信业务经营许可证:粤B2-20120479
©2011-2015 生物帮 All rights reserved.