TraI蛋白在接合性DNA转移的起始和终止过程中具有切割和重新连接单链DNA的活性，其作用如下。

Roles of TraI protein with activities of cleaving and rejoining the single-stranded DNA in both initiation and termination of conjugal DNA transfer.

作者信息

Fukuda H, Ohtsubo E

机构信息

Institute of Molecular and Cellular Biosciences, the University of Tokyo, Japan.

出版信息

Genes Cells. 1997 Dec;2(12):735-51. doi: 10.1046/j.1365-2443.1997.1580356.x.

DOI:10.1046/j.1365-2443.1997.1580356.x

PMID:9544702

Abstract

BACKGROUND

The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood.

RESULTS

We have demonstrated that TraI has the ability to cleave the single-stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5' end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I-footprinting analysis revealed that the TraI bound to the single-stranded DNA of the sbi region. TraI did not cleave the double-stranded DNA fragment, but it did cleave the double-stranded DNA with a single-stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single-stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5' end of one cleaved product with the TraI covalently attached and the 3' end of the other product containing the sbi region.

CONCLUSIONS

Our results demonstrate that the nicking reaction-the initiation reaction of DNA transfer-is actually the cleaving reaction of the single-stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5' end, which is generated by the initiation reaction, with the 3' end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.

摘要

背景

质粒R100编码TraI蛋白，这是接合DNA转移所必需的。TraI具有超螺旋质粒DNA位点特异性和链特异性切口的活性。这种特异性切口的分子机制，被认为是DNA转移的起始反应，目前尚不清楚。

结果

我们已经证明，TraI能够在一个区域内与切口位点（nic）相同的位点切割单链DNA，我们在此将该区域称为sbi。产物包含与切口反应新产生的5'端共价连接的TraI蛋白。切割和切口反应在几乎相同的条件下发生，并且需要sbi区域的存在。DNase I足迹分析表明，TraI与sbi区域的单链DNA结合。TraI不切割双链DNA片段，但它确实能切割在sbi区域有单链DNA部分的双链DNA。KMnO4图谱分析表明，TraI可以使超螺旋DNA中的sbi区域解链以产生单链部分。我们还证明了TraI能够使切割产物重新连接。重新连接反应需要一个切割产物的5'端与共价连接的TraI以及另一个含有sbi区域产物的3'端。