Dutta Arijit, Eckelmann Bradley, Adhikari Sanjay, Ahmed Kazi Mokim, Sengupta Shiladitya, Pandey Arvind, Hegde Pavana M, Tsai Miaw-Sheue, Tainer John A, Weinfeld Michael, Hegde Muralidhar L, Mitra Sankar
Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch (UTMB), Galveston, TX 77555, USA.
Nucleic Acids Res. 2017 Mar 17;45(5):2585-2599. doi: 10.1093/nar/gkw1262.
Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.
微同源性介导的末端连接（MMEJ）是一种易出错的DNA双链断裂（DSB）修复途径，与基因组重排和致癌转化有关；然而，其对辐射诱导的DSB修复的贡献尚未得到明确。我们使用具有3΄-P封闭末端的线性化质粒的环化，模拟X射线诱导的链断裂处的末端，以重现通过MMEJ或非同源末端连接（NHEJ）的DSB修复。对环化质粒的序列分析允许测量MMEJ与NHEJ的相对活性。虽然我们可预测地观察到NHEJ是我们实验中DSB修复的主要途径，但MMEJ在预照射细胞中显著增强，与它们在G2/M期的辐射诱导停滞无关。MMEJ的激活依赖于酪蛋白激酶2（CK2）对XRCC1的磷酸化，增强了XRCC1与末端切除酶MRE11和CtIP的相互作用。MMEJ需要MRE11的内切核酸酶和外切核酸酶活性，这与同源定向DSB修复（HDR）中观察到的情况一样。此外，XRCC1共免疫沉淀复合物（IP）在体外显示出MMEJ活性，照射后显著升高。因此，我们的研究表明，放疗后存活细胞中辐射介导的MMEJ增强可能导致其放射抗性，并且可能成为治疗靶点。
