Center for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, Oregon.
Environ Mol Mutagen. 2013 Oct;54(8):652-8. doi: 10.1002/em.21806. Epub 2013 Aug 1.
DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N(6) -BPDE dA adducts at the second position in the N-ras codon 61(CAA) (61(2) ) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C10 S-BPDE and C10 R-BPDE lesions linked through the N(6) position of adenine in the sequence context N-ras codon 61, position 3 (C10 S-BPDE and C10 R-BPDE at 61(3) ) was carried out in Escherichia coli, and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C10 S-BPDE and C10 R-BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease-deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N-ras 61(3) resulted in truncated products, with the C10 S-BPDE adducts being more blocking than C10 R-BPDE lesions, and termination of synthesis was more pronounced at position 61(3) than at 61(2) for each of the lesions.
由苯并[a]芘-7,8-二氢二醇-9,10-环氧化物（BPDE）加合物在特定位置和立体特异性诱导的 DNA 结构扰动与突变直接相关，导致细胞转化。尽管先前的研究已经确定，在 N-ras 密码子 61（CAA）的第二位（61(2)）含有 N(6)-BPDE dA 加合物的 DNA 的复制仅导致 A 到 G 的转换，但 NMR 分析不仅确定了这种转换突变的结构基础，而且还预测如果加合物位于同一密码子的第三位，则可能会出现扩展的突变谱。为了验证这一预测，在大肠杆菌中进行了含有 C10 S-BPDE 和 C10 R-BPDE 损伤的 DNA 的复制，这些损伤通过腺嘌呤的 N(6)位置连接在 N-ras 密码子 61 的序列背景中，位置 3（C10 S-BPDE 和 C10 R-BPDE 在 61(3)），这些数据显示了广泛的突变谱。除了由两种损伤复制产生的 A 到 G 转换外，C10 S-BPDE 和 C10 R-BPDE 加合物的复制也分别产生了 A 到 C 和 A 到 T 的颠换。使用 Sequenase 2.0 和无外切酶缺陷的大肠杆菌 Klenow 片段和 pol II 进行的单核苷酸掺入分析不仅揭示了高保真度的合成，而且还证明了在特定损伤的对面具有相同的偏好层次结构，与序列背景无关。在 N-ras 61(3)处用两种损伤进行引物延伸测定导致产生截断产物，C10 S-BPDE 加合物比 C10 R-BPDE 损伤更具阻断性，并且对于每种损伤，在 61(3)处的合成终止比在 61(2)处更明显。
