He Mu-Dan, Zhang Feng-Hua, Wang Hua-Lin, Wang Hou-Peng, Zhu Zuo-Yan, Sun Yong-Hua
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; University of the Chinese Academy of Sciences, Beijing 100049, China.
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Mutat Res. 2015 Oct;780:86-96. doi: 10.1016/j.mrfmmm.2015.08.004. Epub 2015 Aug 20.
DNA double-strand break (DSB) repair is of considerable importance for genomic integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are considered as two major mechanistically distinct pathways involved in repairing DSBs. In recent years, another DSB repair pathway, namely, microhomology-mediated end joining (MMEJ), has received increasing attention. MMEJ is generally believed to utilize an alternative mechanism to repair DSBs when NHEJ and other mechanisms fail. In this study, we utilized zebrafish as an in vivo model to study DSB repair and demonstrated that efficient MMEJ repair occurred in the zebrafish genome when DSBs were induced using TALEN (transcription activator-like effector nuclease) or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technologies. The wide existence of MMEJ repair events in zebrafish embryos was further demonstrated via the injection of several in vitro-designed exogenous MMEJ reporters. Interestingly, the inhibition of endogenous ligase 4 activity significantly increased MMEJ frequency, and the inhibition of ligase 3 activity severely decreased MMEJ activity. These results suggest that MMEJ in zebrafish is dependent on ligase 3 but independent of ligase 4. This study will enhance our understanding of the mechanisms of MMEJ in vivo and facilitate inducing desirable mutations via DSB-induced repair.
DNA双链断裂（DSB）修复对于基因组完整性至关重要。同源重组（HR）和非同源末端连接（NHEJ）被认为是参与修复DSB的两种主要的机制不同的途径。近年来，另一种DSB修复途径，即微同源性介导的末端连接（MMEJ），受到了越来越多的关注。一般认为，当NHEJ和其他机制失效时，MMEJ会利用一种替代机制来修复DSB。在本研究中，我们利用斑马鱼作为体内模型来研究DSB修复，并证明当使用转录激活样效应核酸酶（TALEN）或成簇规律间隔短回文重复序列（CRISPR）/Cas9技术诱导DSB时，斑马鱼基因组中会发生高效的MMEJ修复。通过注射几种体外设计的外源性MMEJ报告基因，进一步证明了斑马鱼胚胎中MMEJ修复事件的广泛存在。有趣的是，抑制内源性连接酶4的活性显著增加了MMEJ频率，而抑制连接酶3的活性则严重降低了MMEJ活性。这些结果表明，斑马鱼中的MMEJ依赖于连接酶3，但不依赖于连接酶4。本研究将增进我们对体内MMEJ机制的理解，并有助于通过DSB诱导的修复产生理想的突变。
