Suh M, Ariese F, Small G J, Jankowiak R, Hewer A, Phillips D H
Ames Laboratory-USDOE, Iowa State University 50011, USA.
Carcinogenesis. 1995 Oct;16(10):2561-9. doi: 10.1093/carcin/16.10.2561.
The formation and repair of benzo[a]pyrene diol epoxide-N2-deoxyguanosine adducts (BPDE-N2-dG) in DNA isolated from the skin of mice treated topically with benzo[a]pyrene (BP) was studied by 32P-postlabeling and by low-temperature fluorescence spectroscopy under low resolution and under high resolution fluorescence line narrowing (FLN) conditions. In agreement with earlier studies, total BP-DNA binding reached a maximum at 24 h after treatment (dose: 1 mumol/mouse), then declined rapidly until 4 days after treatment and much more slowly thereafter. An HPLC method was developed which resolved the 32P-postlabeled (-)-trans- from (-)-cis-anti-BPDE-N2-dG, and (+)-trans-from (+)-cis-anti-BPDE-N2-dG. High performance liquid chromatography analysis of the major TLC adduct spot (containing > 80% of the total adducts) obtained by postlabeling BP-modified mouse skin DNA showed that it consisted of a major component that coeluted with (-)-cis-/(+)-trans-anti-BPDE-N2-dG and a minor component that coeluted with (-)-trans-/(+)-cis-anti-BPDE-N2-dG and that the minor component was repaired at a slower rate than the major component. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N2-dG and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N2-dG. In agreement with the 32P-postlabeling results it was observed by fluorescence spectroscopy that the (+)-cis-adducts were repaired more slowly than most other adducts. Moreover, the (+)-trans-adducts exhibited a broad distribution of base-stacked, partially base-stacked and helix-external conformations. Mouse skin DNA samples obtained at early timepoints (2-8 h) after treatment with BP contained substantially more of the 'external' adducts, while samples at later timepoints (24-48 h) contained relatively more adducts in the base-stacked conformation, indicating also that the latter adducts are repaired less readily than the former. The possible biological significance of these novel observations of conformation-dependent rates of DNA adduct repair and their possible dependence on DNA sequence, are discussed.
采用³²P后标记法以及在低分辨率和高分辨率荧光线窄化（FLN）条件下的低温荧光光谱法，研究了用苯并[a]芘（BP）局部处理的小鼠皮肤中分离出的DNA中苯并[a]芘二醇环氧化物-N2-脱氧鸟苷加合物（BPDE-N2-dG）的形成与修复情况。与早期研究一致，处理后24小时（剂量：1 μmol/小鼠）时，总的BP-DNA结合达到最大值，随后迅速下降，直至处理后4天，此后下降更为缓慢。开发了一种高效液相色谱方法，可分离³²P后标记的（-）-反式与（-）-顺式-反-BPDE-N2-dG以及（+）-反式与（+）-顺式-反-BPDE-N2-dG。对通过后标记BP修饰的小鼠皮肤DNA获得的主要薄层层析加合物斑点（占总加合物的80%以上）进行高效液相色谱分析表明，它由一个与（-）-顺式/（+）-反式-反-BPDE-N2-dG共洗脱的主要成分和一个与（-）-反式/（+）-顺式-反-BPDE-N2-dG共洗脱的次要成分组成，且次要成分的修复速度比主要成分慢。完整DNA的低温荧光光谱鉴定出主要加合物为（+）-反式-反-BPDE-N2-dG，次要加合物部分主要由（+）-顺式-反-BPDE-N2-dG组成。与³²P后标记结果一致，荧光光谱观察到（+）-顺式加合物的修复比大多数其他加合物更慢。此外，（+）-反式加合物呈现出碱基堆积、部分碱基堆积和螺旋外部构象的广泛分布。在BP处理后的早期时间点（2 - 8小时）获得的小鼠皮肤DNA样本中，“外部”加合物的含量明显更多，而在后期时间点（24 - 48小时）的样本中，碱基堆积构象的加合物相对较多，这也表明后者加合物的修复比前者更难。讨论了这些关于DNA加合物修复的构象依赖性速率及其可能对DNA序列的依赖性的新观察结果的潜在生物学意义。
