Dahm-Daphi Jochen, Hubbe Petra, Horvath Fruzsina, El-Awady Raafat A, Bouffard Katie E, Powell Simon N, Willers Henning
Laboratory of Experimental Radiation Oncology, Department of Radiotherapy and Radiation Oncology, University Hospital Eppendorf, University of Hamburg, 20246 Hamburg, Germany.
Oncogene. 2005 Mar 3;24(10):1663-72. doi: 10.1038/sj.onc.1208396.
Nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) entails two principal mechanisms: modification of DNA ends prior to ligation (error-prone rejoining) or precise ligation without modification if the DNA ends are complementary (error-free repair). Error-prone rejoining is mutagenic, because it can lead to destruction of coding sequence or to chromosomal aberrations, and therefore must be tightly regulated. Previous studies on the role of the p53 tumor suppressor in the regulation of NHEJ have yielded conflicting results, but a rigorous analysis of NHEJ proficiency and fidelity in a purely chromosomal context has not been carried out. To this end, we created novel repair plasmid substrates that integrate into the genome. DSBs generated by the I-SceI endonuclease within these substrates were repaired by either error-prone rejoining or precise ligation. We found that the expression of wild-type p53 inhibited any repair-associated DNA sequence deletion, including a more than 250-fold inhibition of error-prone rejoining events compared to p53-null cells, while any promoting effect of p53 on precise ligation could not be directly evaluated. The role of p53 in NHEJ appeared to involve a direct transactivation-independent mechanism, possibly restricting DNA end-modification by blocking the annealing of single strands along flanking stretches of microhomology. The inhibition of error-prone rejoining by p53 did not apply to the rejoining of DSBs induced by ionizing radiation. In conclusion, our data suggest that p53 restricts the mutagenic effects of NHEJ without compromising repair proficiency or cell survival, thereby maintaining genomic stability.
DNA双链断裂（DSB）的非同源末端连接（NHEJ）涉及两种主要机制：连接前对DNA末端进行修饰（易错重连），或者如果DNA末端互补则直接精确连接（无错修复）。易错重连具有致突变性，因为它会导致编码序列的破坏或染色体畸变，因此必须受到严格调控。先前关于p53肿瘤抑制因子在NHEJ调控中作用的研究结果相互矛盾，但尚未在纯染色体背景下对NHEJ的效率和保真度进行严格分析。为此，我们构建了可整合到基因组中的新型修复质粒底物。由I-SceI核酸内切酶在这些底物内产生的DSB通过易错重连或精确连接进行修复。我们发现，野生型p53的表达抑制了任何与修复相关的DNA序列缺失，与p53缺失的细胞相比，易错重连事件的抑制倍数超过250倍，而p53对精确连接的任何促进作用无法直接评估。p53在NHEJ中的作用似乎涉及一种直接的非转录激活机制，可能是通过阻止沿着侧翼微同源序列的单链退火来限制DNA末端修饰。p53对易错重连的抑制不适用于电离辐射诱导的DSB重连。总之，我们的数据表明，p53在不损害修复效率或细胞存活的情况下限制了NHEJ的致突变作用，从而维持了基因组稳定性。
