A new functional in vitro cell-free assay to evaluate DNA repair mechanisms

DNA is exposed to endogenous and exogenous agents that are potential causes of several pathological processes; for this reason, eukaryotic cells developed many mechanisms able to control and repair lesions. One of them, Nucleotide Excision Repair (NER) is a highly versatile and complex system by which UV-photolesions, such as cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4) pyrimidone photoproducts (6- 4PPs), are recognized and removed. A key factor, involved in the recognition of chromatin photolesions, is DDB2 (DNA Damaged binding protein 2) thanks to its ability of creating a complex together with DDB1 (UV-DDB complex). Recently, it was demonstrated that DDB2 binds directly PCNA (Proliferating Cell Nuclear Antigen) through a conserved sequence called PIP-box; the disruption of this binding in the mutated form (DDB2PCNA-) induces a delayed DNA damage recognition but also an inefficient DNA repair activation. To better clarify this delay, it was developed a new functional in vitro cell-free system in which repair activity, in isolated nuclei, was evaluated. Its responsiveness was also evaluated with different type of DNA lesions, activating different DNA repair processes, increasing further its applicability. Moreover, the involvement of DDB1 was studied as possible actor when DDB2 loses its function. In the presence of DDB2PCNA- protein, the DNA repair process is inefficient, nevertheless, not completely blocked. For this reason, it was hypothesized a possible ability of DDB1 to bind directly PCNA, when DDB2 is altered or ineffective.