Figure 2.

Approaches to Monitor Resection-Dependent c-NHEJ. (A) An indirect, genetic approach assesses rescue of the Artemis repair defect. Artemis deficiency confers a defect in the slow DSB repair component (detected at times >8 h after X irradiation). Loss or inhibition of factors required for resection relieves this repair defect. This reflects a unique role for Artemis in resolving resection intermediates. (B) A direct approach quantifies pRPA foci formation after α-particle irradiation. pRPA foci numbers are reduced following inhibition or depletion of the described resection factors. We note that Artemis deficiency also reduces pRPA foci numbers after such irradiation. (C) An assay involving an integrated construct monitors rejoining of two closely localised I-SceI DSBs. The construct has a promoter and the CD4 gene separated by an intervening sequence (∼3.2 kbp) that prevents transcription of CD4 [14]. Rejoining can occur resulting in loss of the intervening fragment to generate CD4-expressing cells. Such rejoining frequently involves junctional deletions of 1–20 bp and short microhomologies. We have observed diminished rejoining events in Artemis-deficient cells or cells inhibited or depleted for the described resection factors. Thus, loss of the intervening fragment appears to be promoted by resection. All assays are described in [8]. c-NHEJ, canonical nonhomologous end joining; DSB, double-strand breaks; pRPA, phospho-replication protein A.