Binding of daunomycin to diaminopurine- and/or inosine-substituted DNA.
Binding of daunomycin to diaminopurine- and/or inosine-substituted DNA.
复制标题
道诺霉素与二氨基嘌呤和/或肌苷取代的 DNA 结合。
DOI：
10.1021/bi9716128
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发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
Chaires,JB
中科院分区：
文献类型：
--
作者：
Bailly,C;Suh,D;Waring,MJ;Chaires,JB
抗癌药物道诺霉素与双螺旋DNA的结合已经通过DNA酶I足迹法和荧光滴定法进行了研究，使用一系列聚合酶链式反应（PCR）合成的DNA片段，这些片段包含系统性碱基取代以改变小沟内官能团的分布。160 bptyrTDNA片段构成起始材料。研究了片段，其中（i）肌苷取代鸟苷，（ii）二氨基嘌呤取代腺嘌呤，和（iii）肌苷和二氨基嘌呤分别取代鸟苷和腺嘌呤。这些片段允许2-氨基在小沟中的作用被系统地探索。DNA酶I足迹法实验结果证实道诺霉素优先与正常片段中的5 '（A/T）GC和5'（A/T）CG三联体结合。肌苷取代鸟苷，伴随着小沟中N-2的丢失，削弱了结合亲和力，但没有显着改变与道诺霉素结合相关的序列偏好。然而，通过双取代完全逆转N-2基团的位置，完全改变了柔红霉素的序列偏好，并将其结合从典型的三联体转移到具有5 'IDD基序的三联体。这些结果严格测试并证实了建议的关键作用的柔红霉素的柔红霉素胺部分和9-OH基团在支配结合到优选的网站。在一项平行研究中，研究了与正常tyrT片段的宏观和微观结合，通过使用PCR制备大量长的、确定的DNA序列，实验成为可能。这些实验的结果强调了药物与DNA晶格相互作用的复杂性，并揭示了其对不同结合位点的亲和力的明确异质性。在宏观结合等温线中鉴定并表征了一类高亲和力位点，最可能对应于5 '（A/T）GC和5'（A/T）CG三联体。
The binding of the anticancer drug daunomycin to double-helical DNA has been investigated by DNase I footprinting and fluorescence titration, using a series of polymerase chain reaction (PCR) synthesized DNA fragments that contained systematic base substitutions to alter the disposition of functional groups within the minor groove. The 160 bptyrTDNA fragment constituted the starting material. Fragments in which (i) inosine was substituted for guanosine, (ii) diaminopurine was substituted for adenine, and (iii) both inosine and diaminopurine were substituted for guanosine and adenine, respectively, were studied. These fragments permit the role of the 2-amino group in the minor groove to be systematically explored. The results of DNase I footprinting experiments confirmed that daunomycin binds preferentially to 5‘(A/T)GC and 5‘(A/T)CG triplets in the normal fragment. Substitution of inosine for guanosine, with the concomitant loss of the N-2 in the minor groove, weakened binding affinity but did not dramatically alter the sequence preference associated with daunomycin binding. Complete reversal of the location of the N-2 group by the double substitution, however, completely altered the sequence preference of daunomycin and shifted its binding from the canonical triplets to ones with a 5‘IDD motif. These results have critically tested and confirmed the proposed key roles of the daunosamine moiety and the 9-OH group of daunomycin in dictating binding to preferred sites. In a parallel study, both macroscopic and microscopic binding to the normaltyrTfragment were investigated, experiments made possible by using PCR to prepare large quantities of the long, defined DNA sequence. The results of these experiments underscored the complexity of the interaction of the drug with the DNA lattice and revealed unequivocal heterogeneity in its affinity for different binding sites. A class of high-affinity sites, most probably corresponding to the 5‘(A/T)GC and 5‘(A/T)CG triplets, was identified and characterized in macroscopic binding isotherms.