A paradox in the in vitro end-joining assays

Much work has been focused on the pathways that restore the integrity of the genome after different kinds of lesions, especially double-strand breaks. A classical method to investigate double-strand break repair is the incubation of a DNA substrate with cell-free extracts. In these end-joining assays, the DNA is efficiently ligated by the proteins present in the extract, generating circular molecules and/or multimers. In contrast, using a similar in vitro system, we detected DNA cleavage rather than end ligation. When comparing our results with previous works, a paradox emerges: lower amounts of DNA become multimerized instead of degraded and higher amounts of DNA are degraded rather than multimerized. Here, we have demonstrated that when the DNA/ protein ratio is low enough, the DNA-binding proteins of the nuclear extract protect the DNA substrate, avoiding DNA degradation and vice versa. Therefore, the variation of the DNA/ protein ratio is enough to switch the outcome of the experiment from a DNA cleavage assay to a typical end-joining assay.