Assessing DNA repair is an important endpoint to study the DNA damage response for investigating the biochemical mechanisms of this process and the efficacy of chemotherapy, which often uses DNA damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, they show some limitations mainly due to the lack of chromatin organization. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the correspondent damage-activated cytosolic extracts, containing biotin-16-dUTP, nuclei are able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair reactions. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 or DDB1, stimulated UV-C induced dUTP incorporation. In contrast, a DDB2PCNA- mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different type of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.

A functional in vitro cell-free system for studying DNA repair in isolated nuclei.

Isabella Guardamagna
Investigation
;
Elisabetta Bassi
Investigation
;
Monica Savio
Investigation
;
Paola Perucca
Investigation
;
Ornella Cazzalini
Investigation
;
Ennio Prosperi
Investigation
;
Lucia Anna Stivala
Writing – Review & Editing
2020

Abstract

Assessing DNA repair is an important endpoint to study the DNA damage response for investigating the biochemical mechanisms of this process and the efficacy of chemotherapy, which often uses DNA damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, they show some limitations mainly due to the lack of chromatin organization. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the correspondent damage-activated cytosolic extracts, containing biotin-16-dUTP, nuclei are able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair reactions. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 or DDB1, stimulated UV-C induced dUTP incorporation. In contrast, a DDB2PCNA- mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different type of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11571/1334546
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