Huang J, Logsdon N, Schmieg F I, Simmons D T
Department of Biological Sciences, University of Delaware, Newark 19716-2590, USA.
Oncogene. 1998 Jul 30;17(4):401-11. doi: 10.1038/sj.onc.1201951.
A possible role of p53-dependent transcription in the induction of DNA repair was explored by transfecting a UV-irradiated chloramphenicol acetyl transferase (CAT) reporter plasmid (pRGC.FOS.CAT), containing a minimal FOS promoter driven by a consensus p53 binding site, into a p53 negative-mouse cell line [(10)1]. When a p53-expressing plasmid (pSV.p53) was cotransfected into these cells, CAT expression levels persisted even after prolonged UV irradiation. In comparison, CAT expression from pSV2.CAT, which lacks a p53-responsive element in its SV40 promoter, dropped off much more precipitously after UV irradiation in the absence or presence of WT p53 expression. A similar sharp drop was observed with three other constructs when the reporter gene was under the control of the ras, beta-actin or fos promoter. Mouse cells (A1-5) that constitutively express a temperature-sensitive mutant (135 AV) of mouse p53 also generated, at 32 degrees C, higher levels of enzyme expressed from UV-irradiated pRGC.FOS.CAT than from UV-irradiated pSV2.CAT. The frequency of cyclobutane pyrimidine dimers in UV-irradiated pRGC.FOS.CAT was determined with T4 endo V, and the probability of having an undamaged CAT coding strand was calculated by the Poisson distribution for various times of UV-irradiation. The observed relative CAT expression levels from irradiated pSV2.CAT and pRGC.FOS.CAT in the absence of p53 were consistent with those numbers. These results show that WT p53-mediated transcription directs a resistance of the transcribed DNA to UV inactivation and reactivates the reporter gene. Furthermore, some single point substitution mutants of p53 that maintain a near normal ability to activate transcription had lost their ability to extend CAT gene expression after UV irradiation. Conversely, other mutants with reduced transcriptional activity retained this ability. This indicates that although resistance to UV inactivation is transcriptionally-dependent, these two activities are genetically distinct. These data, taken together, suggest that the transcription of UV-damaged DNA by a p53-dependent process promotes its repair.
通过将含有由共有p53结合位点驱动的最小FOS启动子的紫外线照射的氯霉素乙酰转移酶（CAT）报告质粒（pRGC.FOS.CAT）转染到p53阴性小鼠细胞系[（10）1]中，探索了p53依赖性转录在诱导DNA修复中的可能作用。当将表达p53的质粒（pSV.p53）共转染到这些细胞中时，即使在长时间紫外线照射后，CAT表达水平仍持续存在。相比之下，在其SV40启动子中缺乏p53反应元件的pSV2.CAT，在不存在或存在野生型p53表达的情况下，紫外线照射后CAT表达下降得更为急剧。当报告基因受ras、β-肌动蛋白或fos启动子控制时，用其他三种构建体也观察到类似的急剧下降。组成型表达小鼠p53温度敏感突变体（135AV）的小鼠细胞（A1-5）在32℃时，紫外线照射的pRGC.FOS.CAT表达的酶水平也高于紫外线照射的pSV2.CAT。用T4内切酶V测定紫外线照射的pRGC.FOS.CAT中环丁烷嘧啶二聚体的频率，并通过泊松分布计算在不同紫外线照射时间下具有未受损CAT编码链的概率。在不存在p53的情况下，从照射的pSV2.CAT和pRGC.FOS.CAT观察到的相对CAT表达水平与这些数字一致。这些结果表明，野生型p53介导的转录指导转录的DNA对紫外线失活的抗性并重新激活报告基因。此外，一些保持接近正常转录激活能力的p53单点取代突变体在紫外线照射后失去了延长CAT基因表达的能力。相反，其他转录活性降低的突变体保留了这种能力。这表明，尽管对紫外线失活的抗性是转录依赖性的，但这两种活性在遗传上是不同的。综上所述，这些数据表明，通过p53依赖性过程对紫外线损伤的DNA进行转录可促进其修复。
