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. 2011 Oct 24;286(49):42470–42482. doi: 10.1074/jbc.M111.309252

FIGURE 5.

FIGURE 5.

DSB transcription context affects maintenance of correct end use, but not the degree of end protection during EJ. A, schematic of g4EJ5GFP. Similar to EJ5-GFP, a fragment of an expression cassette including the entire GFP coding sequence is shown separated from the g4 promoter by a 1.8-kb segment flanked by two tandem I-SceI recognition sites. Distal EJ repair of the two tandem I-SceI sites places the g4 promoter directly upstream of rest of the GFP cassette. Shown are the regulator proteins that mediate L-induced transcription (Tx) from the g4 promoter, which are expressed in HEK293-A7 and U2OS-R1. The g4EJ5-GFP reporter was integrated into both of these cell lines. Also shown are diagrams of proximal EJ and distal EJ products resulting from I-SceI/Trex2 expression, both of which involve EJ of the 5′ DSB, which is directly downstream from the g4 promoter. B, the frequency of I-SceI restoration during distal EJ is not affected by transcription context. The g4EJ5-GFP HEK293-A7 cell line was transfected with an I-SceI expression vector in the TxON and TxOFF conditions, and subsequently GFP+ cells were sorted to enrich for distal EJ products that were amplified (primers p2, p4). Shown are representative products digested with I-SceI (S) or uncut (U). C, expression of the I-SceI-Trex2 fusion causes predominantly short deletions to the I-SceI overhang in proximal EJ products, irrespective of DSB transcription context. The g4EJ5GFP U2OS-R1 cell line was transfected with an expression vector for I-SceI-Trex2 fusion in the TxON and TxOFF conditions. Subsequently, I-SceI-resistant proximal EJ products (primers p4 and p5, shown in A) were isolated and cloned for sequencing. Shown are frequencies of sequence junctions for individual clones from the TxON and TxOFF conditions, along with the reference I-SceI site. D, the relative frequency of distal end use is lower in the TxOFF condition, compared with TxON. Cells with the g4EJ5GFP reporter were transfected with I-SceI/Trex2 (separate I-SceI and Trex2 plasmids for HEK293-A7, and the I-SceI-Trex2 fusion for U2OS-R1), under the TxON and TxOFF conditions. Shown are frequencies of distal EJ, proximal EJ, and distal end use from these transfections. Asterisks denote a statistical difference between the two Tx conditions (p < 0.003).