I-SceI-Based Assays to Examine Distinct Repair Outcomes of Mammalian Chromosomal Double Strand Breaks

Abstract

Chromosomal double strand breaks (DSBs) can be repaired by a number of mechanisms that result in diverse genetic outcomes. To examine distinct outcomes of chromosomal DSB repair, a panel of human cell lines has been developed that contain GFP-based reporters with recognition sites for the rare-cutting endonuclease I-SceI. One set of reporters is used to measure DSB repair events that require access to homology: homology-directed repair, homology-directed repair that requires the removal of a nonhomologous insertion, single strand annealing, and alternative end joining. An additional reporter (EJ5-GFP) is used to measure end joining (EJ) between distal DSB ends of two tandem I-SceI sites. These Distal-EJ events do not require access to homology, and thus are distinct from the repair events described above. Indeed, this assay provides a measure of DSB end protection during EJ, via physical analysis of Distal-EJ products to determine the frequency of I-SceI-restoration. The EJ5-GFP reporter can also be adapted to examine EJ of non-cohesive DSB ends, using co-expression of I-SceI with a non-processive 3' exonuclease (Trex2), which can cause partial degradation of the 4 nucleotide 3' cohesive overhangs generated by I-SceI. Such co-expression of I-SceI and Trex2 leads to measurable I-SceI-resistant EJ products that use proximal DSB ends (Proximal-EJ), as well as distal DSB ends (Distal-EJ). Therefore, this co-expression approach can be used to examine the relative frequency of Proximal-EJ versus Distal-EJ, and hence provide a measure of the fidelity of end utilization during repair of multiple DSBs. In this report, the repair outcomes examined by each reporter are described, along with methods for cell culture, transient expression of I-SceI and Trex2, and repair product analysis.