Rao K E, Lown J W
Department of Chemistry, University of Alberta, Edmonton, Canada.
Biochemistry. 1992 Dec 8;31(48):12076-82. doi: 10.1021/bi00163a016.
DNA sequence selectivities in the covalent binding of the antitumor antibiotic saframycins Mx1, Mx3, A, and S have been determined by complementary strand MPE.Fe(II) footprinting and exonuclease III stop assays on two different 545 and 135 base pair long HindIII/RsaI restriction fragments of pBR322 DNA. Saframycins Mx1, Mx3, A, and S recognize primarily 5'-GGG sequences. All four antibiotics also recognize 5'-GGPy sequences, however a cytosine is preferred over a thymine at the 3'-end of this recognition site in all cases. Saframycins Mx1, Mx3 and S, which possess the OH leaving group, also recognize the 5'-CCG sequence, in contrast to saframycin A, which contains the CN leaving group. In contrast, the OH-containing saframycins also recognize the 5'-CTA sequence. Saframycins Mx2, B and C, which lack the critical CN or OH leaving group, do not show any footprints on the restriction fragments examined in this study. The measured binding site size for all four antibiotics is three base pairs. The exonuclease III stop assay independently confirmed the formation of a covalent bond and the strong preference of the antibiotics for 5'-GGG and 5'-GCC sequences. The latter enzyme assay also suggests that the 5'-terminal or central G of the triad binding site is the base to which reversible covalent attachment of the antibiotic takes place.
通过对pBR322 DNA的两个不同的545和135碱基对长的HindIII/RsaI限制性片段进行互补链MPE.Fe(II)足迹分析和核酸外切酶III终止试验，确定了抗肿瘤抗生素沙弗霉素Mx1、Mx3、A和S共价结合中的DNA序列选择性。沙弗霉素Mx1、Mx3、A和S主要识别5'-GGG序列。所有四种抗生素也识别5'-GGPy序列，然而在所有情况下，该识别位点3'-端的胞嘧啶比胸腺嘧啶更受青睐。与含有CN离去基团的沙弗霉素A相比，具有OH离去基团的沙弗霉素Mx1、Mx3和S也识别5'-CCG序列。相比之下，含OH的沙弗霉素也识别5'-CTA序列。缺乏关键的CN或OH离去基团的沙弗霉素Mx2、B和C在本研究中检测的限制性片段上没有显示任何足迹。所有四种抗生素测量的结合位点大小为三个碱基对。核酸外切酶III终止试验独立证实了共价键的形成以及抗生素对5'-GGG和5'-GCC序列的强烈偏好。后一种酶试验还表明，三联体结合位点的5'-末端或中央G是抗生素发生可逆共价连接的碱基。
