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. 2022 Oct 20;50(19):11058–11071. doi: 10.1093/nar/gkac913

Figure 7.

Figure 7.

DSBs repaired in cells expressing K273A Lig4 have characteristics of a-EJ. (A) 293T clones of the indicated genotypes were transfected with Fill-in joining substrate either uncleaved or cleaved with Eco53KI and PspOMI (to generate blunt and 5′ overhangs), or Eco53KI and ApaI(to generate blunt and 3′ overhangs). Cells were assessed for RFP and GFP expression 72 hours later. Student's T test comparing joining rates between Lig4−/− and either Lig4+/+, Lig4+/−, or K273A*/− were performed; ****P < 0.0001; in two-tailed unpaired t test. (B) 293T clones of the indicated genotypes were co-transfected with the alt-VDJ substrate, a ds-RED expression plasmid, and hypermutant RAG expression plasmids. Restoration of the GFP reading frame requires joining via a 9bp region of MH that occurs 10bp from the termini of both coding ends. Cells were assessed for RFP and GFP expression 72 hours after transfection. Student's T test comparing joining rates between Lig4−/− and either Lig4+/+, Lig4+/−, or K273A*/− as well as between Lig4+/+ and either Lig4+/− or K273A*/− were performed; ****P < 0.0001; ***P < 0.001; **P < 0.01; in two-tailed unpaired t test.