High frequency of nucleotide misincorporations upon the processing of double-strand breaks

Base substitutions were detected as a consequence of double-strand break (DSB) repair in plants. The fidelity of processing free DNA ends was analyzed using a stop-codon inactivated beta-glucuronidase (uidA) reporter gene. Circular and linear plasmids carrying the inactive gene were delivered to Nicotiana plumbaginifolia protoplasts or Nicotiana tabacum leaves. Processing of breaks which occurred in close proximity (5-9 bp) to termination codons led to occasional reversions and subsequent gene reactivation. In contrast, the repair of breaks occurring at a greater distance from the stop-codon resulted in a significantly lower number of reversions. The data suggest that the error prone processing of the free ends involves partial degradation and re-synthesis of the DNA repair substrate.