Page J E, Zajc B, Oh-hara T, Lakshman M K, Sayer J M, Jerina D M, Dipple A
Chemistry of Carcinogenesis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702, USA.
Biochemistry. 1998 Jun 23;37(25):9127-37. doi: 10.1021/bi980273v.
The postoligomerization method was used to prepare oligonucleotide 16-mers that contained dAdo or dGuo adducts, derived from trans opening of each enantiomer of the two diastereomeric benzo[a]pyrene 7,8-diol 9,10-epoxides, in two sequence contexts. These 16 oligonucleotides, along with the four corresponding oligonucleotides containing unsubstituted purines, were ligated into single-stranded DNA from bacteriophage M13mp7L2 and transfected into Escherichia coli SMH77. The mutagenic effects of replication past these adducts were then evaluated. The various adduct isomers induced point mutations at different frequencies and with different distributions of mutation types, as was anticipated. However, sequence context had the most substantial effects on mutation frequency. A high frequency of deletions of a single guanine was found in a context where the dGuo adduct was at the 3'-end of a run of five guanines, whereas no single base deletion was found in the other context studied, 5'-CGA-3'. Mutation frequencies in constructs containing dAdo adducts were much higher in a 5'-TAG-3' context (37-58%, depending on the individual isomer) than in a 5'-GAT-3' context (5-20%), and for a given adduct, mutation frequency was up to 10-fold higher in the former sequence than in the latter. These findings indicate that sequence context effects need more thorough evaluation if the goal of understanding the mechanism through which DNA adducts lead to mutation is to be achieved.
采用后寡聚化方法制备了16聚体寡核苷酸，其包含由两种非对映体苯并[a]芘7,8 -二醇9,10 -环氧化物的各对映体的反式开环衍生而来的dAdo或dGuo加合物，处于两种序列背景中。将这16种寡核苷酸，连同四种相应的含有未取代嘌呤的寡核苷酸，连接到来自噬菌体M13mp7L2的单链DNA中，并转染到大肠杆菌SMH77中。然后评估复制经过这些加合物的诱变效应。正如预期的那样，各种加合物异构体以不同频率和不同的突变类型分布诱导点突变。然而，序列背景对突变频率具有最显著的影响。在dGuo加合物位于五个鸟嘌呤的连续序列的3'端的背景中发现了单个鸟嘌呤的高频缺失，而在研究的另一个背景5'-CGA-3'中未发现单个碱基缺失。在5'-TAG-3'背景中（37 - 58%，取决于个体异构体），含有dAdo加合物的构建体中的突变频率比在5'-GAT-3'背景中（5 - 20%）高得多，并且对于给定的加合物，在前一种序列中的突变频率比在后一种序列中高多达10倍。这些发现表明，如果要实现理解DNA加合物导致突变的机制这一目标，序列背景效应需要更全面的评估。
