Abortive ligation intermediate blocks seamless repair of double-stranded breaks

Abstract

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 Mg²⁺-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.

Keywords: Abortive ligation intermediate; Double strand breaks; Seamless repair; Sequential re-ligation; de-adenylation.