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.
Blanco
Miguel G
MG
Departamento de Bioquimica y Biologia Molecular, Facultad de Biologia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, A Coruna, Spain.
Boán
Francisco
F
Gómez-Márquez
Jaime
J
D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D006801:Humans; D066298:In Vitro Techniques; D008957:Models, Genetic; D011995:Recombination, Genetic
D004247:DNA; D011088:DNA Ligases