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. 2017 Mar 20;7:44662. doi: 10.1038/srep44662

Figure 2. DEK is required for HR DSB repair.

Figure 2

(a) Schematic of two episomal HR reporter plasmids. After co-transfection with an I-SceI plasmid, the mutant GFP gene (light green) site indicated by double arrows is cleaved. The circle represents the corresponding region of homology to the I-SceI site, and the X represents gene truncations. Expression of functional GFP (dark green) is dependent on HR repair of the cleaved plasmid. (b) GFP+ FACs quantification of repair frequency of the constructs in (A). (Mann-Whitney test, n = 12 for HR-EGFP/5′EGFP and n = 5 for pHPRT-DRGFP, mean ± SEM) (c) Schematic of the 6xTer vector. Tus-Ter binding induces replication fork stalling at the 6xTer array (red hourglass) while I-SceI creates a DSB at the location indicated by the double arrows. HR repair triggered by I-SceI or Tus expression results in short tract gene conversion (STGC, GFP+ RFP−), or long-tract gene conversion (LTGC) through the duplication and proper alignment of two synthetic RFP exons allowing RFP expression by alternative mRNA splicing (GFP+ RFP+). (d) Western blot analysis of 6xTer mES cells after siDek treatment. (e) Quantification of total HR repair (STGC + LTGC) and individual STGC and LTGC repair FACs following I-SceI transfection. (Unpaired Welch’s t test, n = 6, mean ± SEM) (f) STGC and LTGC HR repair following Tus transfection. (Unpaired Welch’s t test, n = 6, mean ± SEM).