A3G mediates deletional repair of a persistent ISceI-induced DSB. (A) Scheme of the DR-GFP HR reporter assay.²³  Repair of ISceI-induced DSB by HR reconstitutes the expression of functional GFP. (B) HRind cells were transfected with an EV, A3G (WT), or A3G W285A expression plasmids and induced with TA for 52 hours. GFP expression was measured by FACS. Transfection yield was 55% to 60%, as determined by cotransfection with DsRed expression plasmid. Expression of A3G and A3G W285A was evaluated by Western blot (top). Values represent mean ± SD from 3 independent experiments. *P = .007 (unpaired t test). (C) ISceI-expressing lentiviral vector containing long terminal repeats (LTRs), internal ribosomal entry site (IRES), and ISceI target sequence (I) adjacent to GFP. (D) H9 or SupT1 cells were infected with lentiviruses containing the ISceI vector or mock and assessed 48 hours later for GFP⁺ cells by FACS (0 hours). Cells were sorted again 52 hours after induction of ISceI with TA (52 hours). Values represent mean ± SD from 3 independent experiments. (E) Genomic DNA was extracted from mock- or ISceI lentivirus-infected H9 or SupT1 52 hours after ISceI induction with TA. *Analysis of PCR amplification of a 5900-bp fragment using vector specific primers was performed by a Bioanalyzer. DNA marker (M) sizes are indicated (kb).