Figure_4.tif (443.96 kB)
Download file

Expression of the Trex2 exonuclease promotes the formation of Xrcc4-dependent I-SceI-resistant EJ products.

Download (0 kB)
figure
posted on 16.10.2009, 00:00 authored by Nicole Bennardo, Amanda Gunn, Anita Cheng, Paul Hasty, Jeremy M. Stark

(A) Shown are primers for EJ5-GFP that are used to analyze proximal-EJ at the 5′ and 3′ I-SceI sites (shown as 5′S and 3′S, respectively), as well as Distal-EJ. (B) Expression of the Trex2 exonuclease promotes the formation of I-SceI-resistant EJ products between proximal DSB ends at the 3′ I-SceI site in EJ5-GFP. WT ES cells with EJ5-GFP were transfected with an I-SceI expression vector (S) along with a Trex2 expression vector (Trex2), an exonuclease-deficient mutant of Trex2 (Trex2-H188A), or EV. Shown are amplification products from these transfections using the primers p1 and p2, which were either left uncut (U) or were cut with I-SceI (S). (C) Trex2 expression also promotes I-SceI-resistant EJ products in Trex2null cells. Analysis was performed on Trex2null cells with the EJ5-GFP reporter as described in B. (D) Trex2 expression promotes I-SceI-resistant EJ products at the 5′ I-SceI site of EJ5-GFP. Shown are amplification products using the primers p3 and p4, using the same transfection conditions and annotation as described in B. (E) Trex2 expression promotes I-SceI-resistant Distal-EJ products. Shown are amplification products from sorted GFP+ cells derived from the transfections shown in C, using primers p3 and p2, with the same annotation shown in B. (F) EJ products via Trex2 show deletion of segments of the I-SceI 3′ overhang (underlined). The I-SceI-resistant products shown in C were cloned, and 11 individual clones were sequenced. Shown are the sequences of these clones, where the numerator in parenthesis depicts the number of times a given sequence was identified. An asterisk denotes the one clone with evidence of microhomology (1 nt., A in bold). (G) I-SceI-resistant proximal EJ products via Trex2 are dependent on Xrcc4. Analysis of the effect on Trex2 expression on the 3′ I-SceI site of the EJ5-GFP reporter was performed on Xrcc4−/− cells as described in B.

History

Usage metrics

Licence

Exports