MboII限制性内切核酸酶与DNA的相互作用。

Interaction of the MboII restriction endonuclease with DNA.

作者信息

Sektas M, Kaczorowski T, Podhajska A J

机构信息

Department of Microbiology, University of Gdańsk, Poland.

出版信息

Gene. 1995 May 19;157(1-2):181-5. doi: 10.1016/0378-1119(94)00742-b.

DOI:10.1016/0378-1119(94)00742-b

PMID:7607488

Abstract

The binding of the MboII restriction endonuclease (R.MboII; ENase) to DNA containing its recognition site was investigated using a mobility shift assay. R.MboII forms specific, stable and immunodetectable complexes with its canonical target sequence. The association constant (Ka) of R.MboII was calculated to be 2.8 x 10(9)/M, and is about 10(4)-fold higher than the Ka value for non-specific binding. Based on results obtained after sedimentation of the R.MboII-DNA complex in a glycerol gradient and measurement of the retardation of the complexes in polyacrylamide gels, we conclude that specific binding to the canonical sequence involves a monomer of R.MboII. DNase I footprinting has shown that the enzyme covers 16 nucleotides of DNA on the 5'-GAAGA-3' strand.

摘要

利用迁移率变动分析法研究了MboII限制性内切酶（R.MboII；ENase）与含有其识别位点的DNA的结合情况。R.MboII与其典型靶序列形成特异性、稳定且可免疫检测的复合物。R.MboII的缔合常数（Ka）经计算为2.8×10⁹/M，比非特异性结合的Ka值高约10⁴倍。基于在甘油梯度中对R.MboII-DNA复合物进行沉降以及测量聚丙烯酰胺凝胶中复合物的阻滞情况后获得的结果，我们得出结论，与典型序列的特异性结合涉及R.MboII的一个单体。DNase I足迹分析表明，该酶覆盖了5'-GAAGA-3'链上16个核苷酸的DNA。