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Papers In Press, published online ahead of print April 15, 2004
J. Biol. Chem, 10.1074/jbc.M402832200
Submitted on March 12, 2004
Revised on April 6, 2004
Accepted on April 15, 2004

A paradox in the in vitro end-joining assays

Miguel G. Blanco, Francisco Boan, and Jaime Gomez-Marquez

Departamento de Bioquimica y Biologia Molecular, Universidad de Santiago de Compostela, Santiago de Compostela, Galicia 15706

Corresponding Author: bnjgm{at}usc.es

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 demonstrate 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.


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