Konakahara T, Wurdeman R L, Gold B
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68105.
Biochemistry. 1988 Nov 15;27(23):8606-13. doi: 10.1021/bi00423a015.
The synthesis and characterization of an N-methyl-N-nitrosourea (MNU) analogue that is covalently linked to a methidium nucleus is described. At 37 degrees C in pH 8.0 buffer 9 hydrolyzes via pseudo-first-order kinetics, with a calculated t1/2 = 77 min. By use of polyacrylamide sequencing gels the formation of piperidine-labile N7-methylguanine adducts from the reaction of 9 and MNU with 5'-32P-end-labeled DNA restriction fragments is reported. DNA methylation by 9 in 10 mM Tris buffer is enhanced with increasing ionic strength (50-200 mM NaCl), which contrasts to the inhibition of MNU-induced cleavage with increasing salt. In addition, 9 methylates all G sites equally, while MNU shows a clear preference for d(G)n (n greater than or equal to 3) runs and an asymmetrical methylation pattern within these G-rich regions. The results are discussed in terms of the delivery of the MNU moiety to the DNA target by a non-sequence-specific intercalation process and the subsequent hydrolytic generation of a nondiffusible alkylating intermediate.
描述了一种与甲啶核共价连接的N-甲基-N-亚硝基脲（MNU）类似物的合成与表征。在37℃、pH 8.0缓冲液中，9通过准一级动力学水解，计算得到的t1/2 = 77分钟。通过使用聚丙烯酰胺测序凝胶，报道了9和MNU与5'-32P末端标记的DNA限制片段反应形成哌啶不稳定的N7-甲基鸟嘌呤加合物。在10 mM Tris缓冲液中，9引起的DNA甲基化随离子强度增加（50 - 200 mM NaCl）而增强，这与MNU诱导的切割随盐浓度增加而受到抑制形成对比。此外，9对所有G位点的甲基化程度相同，而MNU明显偏好d(G)n（n≥3）序列，并且在这些富含G的区域内呈现不对称的甲基化模式。根据通过非序列特异性嵌入过程将MNU部分递送至DNA靶点以及随后水解产生不可扩散的烷基化中间体来讨论这些结果。
