Figure 1.

Quantitative assay for the repair of cDSB in cultured mammalian cells. (A) Flow chart of the assay. Cells were transfected with a DNA mixture containing a modified linear plasmid with an abasic site on a 5′-overhang (LP41, kan^R), a normalizing intact plasmid (pSA26, cm^R) and a carrier plasmid (pUC18, amp^R). In parallel, cells were transfected with a control mixture containing a non-modified linear plasmid with a 5′-overhang but no abasic site (LP40, kan^R). Cells were incubated to allow repair, after which the plasmids were extracted and elctroporated into indicator E. coli cells. Finally, the bacteria cells were seeded in parallel on LB plates containing kanamycin or chloramphenicol. The relative repair of the cDSB relative to the sDSB was deduced from the colonies count, as described in the text. LP40 and LP41 descendent plasmids were recovered from the bacteria colonies, and subjected to DNA sequence analysis at the vicinity of the original break point. (B) The break points of of the linear plasmids carrying a cDSB (LP41) and a sDSB (LP40).