Diekmann S, McLaughlin L W
Max-Planck-Institut für biophysikalische Chemie, Göttingen, F.R.G.
J Mol Biol. 1988 Aug 20;202(4):823-34. doi: 10.1016/0022-2836(88)90561-x.
The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (kcat/Km), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein-nucleic acid interactions observed for the EcoRI restriction endonuclease.
连接含有EcoRI识别位点的十聚脱氧核苷酸会形成一系列多聚体，基于在聚丙烯酰胺凝胶中观察到的异常凝胶迁移现象，这些多聚体似乎是弯曲的。根据观察到的迁移异常情况，识别序列中存在的DNA弯曲程度可通过对嘌呤2位和6位官能团的修饰而改变。发生在B-DNA螺旋小沟中的鸟嘌呤2-氨基的缺失，在增加观察到的DNA弯曲方面最为有效。相反，将一个氨基从大沟转移到小沟会消除弯曲。嘌呤6位的环外基团也会调节DNA弯曲，当将氨基换成羰基或质子取代基时，观察到弯曲程度降低。许多修饰序列的内切酶切割反应动力学参数的差异，是大沟中官能团修饰的结果，这些官能团在催化过程中可能与内切酶接触。然而，有两个例子中，表征蛋白质-核酸相互作用的观察到的特异性常数（kcat/Km）显著降低，很难用这种官能团接触来解释。在这些情况下，更有可能的是官能团修饰通过调节双链DNA螺旋结构来影响内切酶与DNA相互作用的效率。在这两个例子中，都对小沟取代基进行了修饰。与天然识别位点相比，其中一个例子中DNA弯曲程度显著增加，另一个例子中则降低。结果表明，DNA螺旋轴的弯曲是d(GAATTC)序列的固有特性，有助于优化EcoRI限制性内切酶所观察到的蛋白质-核酸相互作用。
