In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex.
In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex.
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DNA 硫代磷酸酯修饰的体外分析揭示多蛋白复合物对底物的识别
DOI：
10.1038/srep12513
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发表时间：
2015-07-27
影响因子：
4.6
通讯作者：
You D
中科院分区：
文献类型：
--
作者：
Cao B;Zheng X;Cheng Q;Yao F;Zheng T;Ramesh Babu I;Zhou H;Dedon P;You D
各种各样的原核生物具有DNA修饰组成的序列特异性硫代磷酸酯（PT）插入一个五基因簇的成员。最近的基因组作图研究揭示了PT修饰的两个不寻常的特征：短的共有序列和细菌群体中特定基因组位点的部分修饰。为了更好地理解这些特征的基础PT修饰的靶选择机制，我们表征了沙门氏菌细胞提取物系统中称为DptC、D和E的PT修饰酶的底物识别。结果表明，双链寡核苷酸在体外发生了PT的从头修饰，其修饰模式与体内相同，即：例如，GpsAAC/GpsTTC基序。出乎意料的是，在这些体外分析中，我们观察到GAAC/GTTC基序侧翼序列对PT修饰没有显著影响，而PT也发生在体内不能修饰的GAAC/GTTC基序中。半PT DNA也作为PT修饰酶的底物，但不是单链DNA。然后发现PT修饰酶作为一个大的蛋白质复合物发挥作用，所有三个亚基都呈四聚体构象。这项研究首次证明了PT修饰酶作为一个大的蛋白质复合物在体外DNA PT修饰。
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 selection 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.