Hartley J A, Forrow S M, Souhami R L
Department of Oncology, University College, London, U.K.
Biochemistry. 1990 Mar 27;29(12):2985-91. doi: 10.1021/bi00464a014.
Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increased ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. For L-Pam and UM, increased ionic strength and the cationic DNA affinity binders dose dependently inhibited the alkylation. QM alkylation was less inhibited by salt (100 mM NaCl), ethidium (10 microM), and spermine (10 microM). Distamycin A and netropsin (100 microM) gave an enhancement of overall QM alkylation. More interestingly, the pattern of guanine N7-alkylation was qualitatively altered by ethidium bromide, distamycin A, and netropsin. The result differed with both the nitrogen mustard (L-Pam less than UM less than QM) and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.
在用氮芥等化疗烷基化剂处理后，DNA序列中的鸟嘌呤之间的烷基化强度存在很大差异，并且连接到反应基团上的取代基会对反应产生明显的序列偏好。为了进一步了解决定烷基化反应序列选择性的结构和静电因素，通过改进用于DNA测序的鸟嘌呤特异性化学切割技术，研究了增加离子强度、嵌入剂溴化乙锭、AT特异性小沟结合剂放线菌素A和纺锤菌素以及多胺精胺对L-苯丙氨酸氮芥（L-Pam）、尿嘧啶氮芥（UM）和喹吖因氮芥（QM）引起的鸟嘌呤N7-烷基化的影响。对于L-Pam和UM，增加离子强度和阳离子DNA亲和结合剂剂量依赖性地抑制烷基化。QM烷基化受盐（100 mM NaCl）、溴化乙锭（10 μM）和精胺（10 μM）的抑制较小。放线菌素A和纺锤菌素（100 μM）使QM的总体烷基化增强。更有趣的是，溴化乙锭、放线菌素A和纺锤菌素使鸟嘌呤N7-烷基化模式发生了定性改变。结果因氮芥（L-Pam＜UM＜QM）和所用阳离子剂的不同而有所差异。这种导致烷基化位点增强和抑制的效应在纺锤菌素和放线菌素A的情况下最为显著，它们彼此不同。DNA足迹分析表明，在DNA小沟中与AT序列的选择性结合可对DNA大沟中的烷基化模式产生远程影响。
