Fig. 1: SSA-mediated gene targeting becomes prominent when DSB repair by HR or EJ is deficient.
From: Characterization and regulation of cell cycle-independent noncanonical gene targeting
a Schematic of gene-targeting assay to calculate gene-targeting efficiency, targeted integration (TI) frequency, and random integration (RI) frequency. Puror, puromycin-resistance gene; 6TG, 6-thioguanine. b Scheme of gene targeting assay at the HPRT locus. Homologous integration of p8.9HPRT-2A-Puro in 6TG-resistant (i.e., HPRT-deficient) cells were confirmed by PCR analysis using three different primer sets (A/B, C/D, and E/F). Shown are the results of three 6TG-resistant clones (lanes 1−3) and a 6TG-sensitive HPRT + clone (lane 4) (n = 1; note that one clone (lane 3) is a random integrant harboring a spontaneous HPRT mutation.) M, HindIII-digested λDNA. c Impact of Rad51/Rad52 inhibition on gene targeting in wild-type and RAD54–/–RAD54B–/– cells. Cells were transfected with linearized p8.9HPRT-2A-Puro, and treated with Rad51 inhibitor (B02 or RI-2) or Rad52 inhibitor (DOPA or AICAR) prior to replating. Data shown are the mean ± s.d. (n = 3 for Rad51i; n = 5 for Rad52i). d Impact of dual inhibition of Rad51 and Rad52 on TI frequency. p8.9HPRT-2A-Puro-transfected cells were treated with either or both of Rad51/Rad52 inhibitors prior to replating. Symbols are as in (c). Data shown are the mean ± s.d. (n = 3 for wild-type and RAD54–/–RAD54B–/–; n = 4 for LIG4–/–POLQ–/–). Statistical significance in (c) and (d) was determined by two-sided Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Source data are provided as a Source Data file. e Schematic representation of targeted and random integration (TI and RI) of targeting vector. RI occurs in a homology-independent manner and relies on either Ku/LIG4-dependent NHEJ or POLθ-dependent TMEJ, while TI is homology-dependent. In addition to HR that relies on Rad51/Rad54/Rad54B, a noncanonical TI mechanism has been uncovered that is dependent on Rad52 but not Rad51. It should be noted that TI and RI are highly infrequent; neither TI nor RI does not occur in >99.99% of transfected cells.
