Li Xuegang, Jin Jiacheng, Xu Wenxuan, Wang Mingdao, Liu Liangwei
The Life Science College, Henan Agricultural University, Zhengzhou 450002, China.
The Life Science College, Henan Agricultural University, Zhengzhou 450002, China; The Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Henan Agricultural University, Zhengzhou 450002, China.
Int J Biol Macromol. 2022 Jun 1;209(Pt A):1498-1503. doi: 10.1016/j.ijbiomac.2022.04.098. Epub 2022 Apr 22.
Because indel results in frame-shift mutations, seamless repair of double-stranded break (DSB)s plays a pivotal role in synthetic biology, molecular biology, and genome integrity. However, DSB repair is not well documented. T4 DNA ligase (T4lig) served to ligate intra-molecularly a zero bp break-apart DSB linear plasmid DNA pET22b(28a)-xylanase. An ATP T4lig ligation reaction joined one single-stranded break (SSB) into a phosphodiester-bond, whereas the opposite SSB into an abortive ligation intermediate blocking the DSB sequential repair. The intermediate proved to be fluorescent Cy5-AMP-SSB by a T4lig ligation reaction in the aid of Alexa Fluor 647 ATP having Cy5-AMP fluorescence. The fluorescent Cy5-AMP-SSB was de-adenylated into SSB by an ATP-free T4lig or Mg-free T4ligL159L reaction. The de-adenylated SSB was re-joined into another phosphodiester-bond by a sequential ATP T4lig re-ligation reaction. Thereby, DSB repair proceeds an abortive ligation, a reverse de-adenylation, and a sequential re-ligation reaction. The result has a potential usage in synthetic biology, molecular biology, and cancer-curing.
由于插入缺失会导致移码突变，双链断裂（DSB）的无缝修复在合成生物学、分子生物学和基因组完整性中起着关键作用。然而，关于DSB修复的文献记载并不充分。T4 DNA连接酶（T4lig）用于分子内连接零碱基对断裂的DSB线性质粒DNA pET22b(28a)-木聚糖酶。ATP T4lig连接反应将一个单链断裂（SSB）连接成磷酸二酯键，而相对的SSB则连接成阻止DSB顺序修复的无效连接中间体。借助具有Cy5-AMP荧光的Alexa Fluor 647 ATP，通过T4lig连接反应证明该中间体为荧光Cy5-AMP-SSB。荧光Cy5-AMP-SSB通过无ATP的T4lig或无镁的T4ligL159L反应脱腺苷酸化成为SSB。脱腺苷酸化的SSB通过顺序ATP T4lig重新连接反应重新连接成另一个磷酸二酯键。因此，DSB修复通过无效连接、反向脱腺苷酸化和顺序重新连接反应进行。该结果在合成生物学、分子生物学和癌症治疗中具有潜在用途。
