Cunniffe Siobhan M T, Lomax Martine E, O'Neill Peter
Medical Research Council, Radiation and Genome Stability Unit, Harwell, Didcot, Oxfordshire OX11 0RD, UK.
DNA Repair (Amst). 2007 Dec 1;6(12):1839-49. doi: 10.1016/j.dnarep.2007.07.003. Epub 2007 Aug 20.
Ionizing radiation induces clustered DNA damaged sites, defined as two or more lesions formed within one or two helical turns of the DNA through passage of a single radiation track. It is now established that clustered DNA damage sites are found in cells and present a challenge to the repair machinery of the cell but to date, most studies have investigated the effects of bi-stranded lesions. A subset of clustered DNA damaged sites exist in which two or more lesions are present in tandem on the same DNA strand. In this study synthetic oligonucleotides containing an AP site 1, 3 or 5 bases 5' or 3' to 8-oxo-7,8-dihydroguanine (8-oxoG) on the same DNA strand were synthesized as a model of a tandem clustered damaged sites. It was found that 8-oxoG retards the incision of the AP site by exonuclease III (Xth) and formamidopyrimidine DNA glycosylase (Fpg). In addition the rejoining of the AP site by xrs5 nuclear extracts is impaired by the presence of 8-oxoG. The mutation frequency arising from 8-oxoG within a tandem clustered site was determined in both wild type and mutant E. coli backgrounds. In wild-type, nth, fpg and mutY null E. coli, the mutation frequency is slightly elevated when an AP site is in tandem to 8-oxoG, compared with when 8-oxoG is present as a single lesion. Interestingly, in the double mutant mutY/fpg null E. coli, the mutation frequency of 8-oxoG is reduced when an AP site is present in tandem compared with when 8-oxoG is present as a single lesion. This study demonstrates that tandem lesions can present a challenge to the repair machinery of the cell.
电离辐射会诱导形成簇状DNA损伤位点，其定义为通过单个辐射径迹在DNA的一或两个螺旋圈内形成的两个或更多个损伤。现已确定，簇状DNA损伤位点存在于细胞中，并对细胞的修复机制构成挑战，但迄今为止，大多数研究都调查了双链损伤的影响。存在一部分簇状DNA损伤位点，其中两个或更多个损伤在同一DNA链上串联存在。在本研究中，合成了含有与同一DNA链上8-氧代-7,8-二氢鸟嘌呤（8-氧代鸟嘌呤，8-oxoG）的5'或3'端相隔1、3或5个碱基的AP位点的合成寡核苷酸，作为串联簇状损伤位点的模型。研究发现，8-氧代鸟嘌呤会阻碍核酸外切酶III（Xth）和甲酰胺嘧啶DNA糖基化酶（Fpg）对AP位点的切割。此外，8-氧代鸟嘌呤的存在会损害xrs5核提取物对AP位点的重新连接。在野生型和突变型大肠杆菌背景下，均测定了串联簇状位点内8-氧代鸟嘌呤引起的突变频率。在野生型、nth、fpg和mutY基因缺失的大肠杆菌中，与8-氧代鸟嘌呤作为单个损伤存在时相比，当AP位点与8-氧代鸟嘌呤串联时，突变频率略有升高。有趣的是，在mutY/fpg双突变基因缺失的大肠杆菌中，与8-氧代鸟嘌呤作为单个损伤存在时相比，当存在串联的AP位点时，8-氧代鸟嘌呤的突变频率降低。本研究表明，串联损伤可能会对细胞的修复机制构成挑战。
