Hailer-Morrison M Katie, Kotler J Michelle, Martin Brooke D, Sugden Kent D
Department of Chemistry, The University of Montana, Room CP304, Missoula, Montana 59812, USA.
Biochemistry. 2003 Aug 19;42(32):9761-70. doi: 10.1021/bi034546k.
A number of common promoter elements that drive transcription of redox sensitive genes have runs of guanines in their transcription factor recognition sequence. A paradox exists insomuch that the same guanine runs necessary for transcription factor recognition are thermodynamically prone to oxidative modification, potentially altering the binding affinity of transcription factors. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) is a common oxidative modification of guanine that is generated by a variety of metals and reactive oxygen species. We have used the p50 subunit of the NF-kappaB transcription factor to show that oxidation of guanine to 8-oxo-dG at sites critical for protein recognition impacts transcription factor binding affinity differently depending upon the site of oxidation. It can be argued that the impact of such oxidation will be minimal in repair proficient cells. Therefore, we have developed an assay to assess the ability of these lesions to be shielded by transcription factor binding from recognition and repair by base excision repair (BER) enzymes. In this study, 8-oxo-dG was substituted for guanine at sites G(1)-G(4) in the NF-kappaB sequence 5'-d(AGTTGAG(1)G(2)G(3)G(4)ACTTTCCCAGCC)-3'. We have observed that substitution of 8-oxo-dG at the G(1) site increases p50 binding affinity by approximately 2.5-fold compared to that of the unmodified DNA sequence, while substitution at G(3) reduces the binding affinity by approximately 4-fold. Substitution of 8-oxo-dG at the G(2) and G(4) sites had a minimal impact on p50 binding affinity. Both Escherichia coli fapy glycosylase (Fpg) and human 8-oxo-DNA glycosylase (hOGG1) recognized and cleaved 8-oxo-dG at all four sites within the promoter element. The addition of the p50 transcription factor shielded these lesions from cleavage by the glycosylase in a manner that correlated with the binding affinities of p50 for the different modified sites. These data imply that lesion formation in DNA response elements can modulate gene transcription during oxidative events and that protein binding to these modified sites may allow these lesions to persist on a time scale that impacts global cellular gene transcription.
许多驱动氧化还原敏感基因转录的常见启动子元件在其转录因子识别序列中存在鸟嘌呤串联。存在一个悖论，即转录因子识别所必需的相同鸟嘌呤串联在热力学上易于发生氧化修饰，这可能会改变转录因子的结合亲和力。7,8-二氢-8-氧代-2'-脱氧鸟苷（8-氧代-dG）是鸟嘌呤常见的氧化修饰产物，由多种金属和活性氧产生。我们利用核因子κB转录因子的p50亚基表明，在对蛋白质识别至关重要的位点将鸟嘌呤氧化为8-氧代-dG，对转录因子结合亲和力的影响因氧化位点而异。可以认为，这种氧化在修复能力强的细胞中的影响将最小。因此，我们开发了一种检测方法，以评估这些损伤被转录因子结合所屏蔽，从而免受碱基切除修复（BER）酶识别和修复的能力。在本研究中，在核因子κB序列5'-d(AGTTGAG(1)G(2)G(3)G(4)ACTTTCCCAGCC)-3'的G(1)-G(4)位点用8-氧代-dG替代鸟嘌呤。我们观察到，与未修饰的DNA序列相比，在G(1)位点替换8-氧代-dG可使p50结合亲和力增加约2.5倍，而在G(3)位点替换则使结合亲和力降低约4倍。在G(2)和G(4)位点替换8-氧代-dG对p50结合亲和力的影响最小。大肠杆菌fapy糖基化酶（Fpg）和人8-氧代-DNA糖基化酶（hOGG1）在启动子元件内的所有四个位点都能识别并切割8-氧代-dG。添加p50转录因子以与p50对不同修饰位点的结合亲和力相关的方式屏蔽这些损伤，使其免受糖基化酶的切割。这些数据表明，DNA反应元件中的损伤形成可在氧化事件期间调节基因转录，并且蛋白质与这些修饰位点的结合可能使这些损伤在影响全局细胞基因转录的时间尺度上持续存在。
