Walter R B, Pierce J, Case R, Tang M S
University of Texas System Cancer Center, Science Park-Research Division, Smithville 78957.
J Mol Biol. 1988 Oct 20;203(4):939-47. doi: 10.1016/0022-2836(88)90119-2.
The binding of the anti-tumor antibiotic anthramycin to a defined linear DNA fragment was investigated using both exonuclease III and lambda exonuclease. We show that most of the guanine residues are reactive toward anthramycin; however, several guanine residues showed preferential reactivity for the drug. Using purified UVRA, UVRB and UVRC proteins we present evidence that these three proteins in concert are able to recognize and produce specific strand cleavage flanking anthramycin-DNA adducts. The cleavage of anthramycin adducts by UVRABC nuclease is specific and results in strand breaks at five or six bases 5' and three or four bases 3'-flanking an adduct. At some guanine residues single incisions were observed only on one side of the adduct. The 5' strand breaks observed often occurred as doublet bands on sequencing gels, indicating plasticity in the site of 5' cleavage whereas the 3' cleavage did not show this effect. When DNA fragments modified with elevated levels of anthramycin were used as substrates the activity of the UVRABC nuclease toward the anthramycin adducts decreased. Possible mechanisms for the recognition and specific cleavage of the helix-stabilizing anthramycin DNA adduct and other helix destabilizing lesions by the UVRABC nuclease are discussed.
使用核酸外切酶III和λ核酸外切酶研究了抗肿瘤抗生素氨茴霉素与特定线性DNA片段的结合。我们发现大多数鸟嘌呤残基对氨茴霉素具有反应性；然而，有几个鸟嘌呤残基对该药物表现出优先反应性。使用纯化的UVRA、UVRB和UVRC蛋白，我们提供证据表明这三种蛋白协同作用能够识别并在氨茴霉素-DNA加合物两侧产生特异性链切割。UVRABC核酸酶对氨茴霉素加合物的切割具有特异性，导致在加合物5'端的五或六个碱基和3'端的三或四个碱基处发生链断裂。在一些鸟嘌呤残基处，仅在加合物的一侧观察到单一切口。在测序凝胶上观察到的5'链断裂经常以双峰带出现，表明5'切割位点具有可塑性，而3'切割未显示出这种效应。当使用用高水平氨茴霉素修饰的DNA片段作为底物时，UVRABC核酸酶对氨茴霉素加合物的活性降低。讨论了UVRABC核酸酶识别和特异性切割稳定螺旋的氨茴霉素DNA加合物以及其他破坏螺旋的损伤的可能机制。
