title: DDB2 association with PCNA is required for its degradation after UV-induced DNA damage.
abstract: DDB2 is a protein playing an essential role in the lesion recognition step of the global genome sub-pathway of nucleotide excision repair (GG-NER) process. Among the proteins involved in the DNA damage response, p21(CDKN1A) (p21) has been reported to participate in NER, but also to be removed by proteolytic degradation, thanks to its association with PCNA. DDB2 is involved in the CUL4-DDB1 complex mediating p21 degradation; however, the direct interaction between DDB2, p21 and PCNA has been never investigated. Here, we show that DDB2 co-localizes with PCNA and p21 at local UV-induced DNA-damage sites, and these proteins co-immunoprecipitate in the same complex. In addition, we provide evidence that p21 is not able to bind directly DDB2, but, to this end, the presence of PCNA is required. Direct physical association of recombinant DDB2 protein with PCNA is mediated by a conserved PIP-box present in the N-terminal region of DDB2. Mutation of the PIP-box resulted in the loss of protein interaction. Interestingly, the same mutation, or depletion of PCNA by RNA interference, greatly impaired DDB2 degradation induced by UV irradiation. These results indicate that DDB2 is a PCNA-binding protein, and that this association is required for DDB2 proteolytic degradation.
authors.affiliation: Dipartimento di Medicina Molecolare; Unità di Immunologia e Patologia Generale; Università di Pavia; Pavia, Italy.
authors.forename: Ornella
authors.initials: O
authors.lastname: Cazzalini
authors.affiliation: Dipartimento di Medicina Molecolare; Unità di Immunologia e Patologia Generale; Università di Pavia; Pavia, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Perucca
authors.affiliation: Dipartimento di Medicina Molecolare; Unità di Immunologia e Patologia Generale; Università di Pavia; Pavia, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Mocchi
authors.affiliation: Dipartimento di Medicina Molecolare; Unità di Immunologia e Patologia Generale; Università di Pavia; Pavia, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Sommatis
authors.affiliation: Istituto di Genetica Molecolare (IGM) del CNR; Pavia, Italy.
authors.forename: Ennio
authors.initials: E
authors.lastname: Prosperi
authors.affiliation: Dipartimento di Medicina Molecolare; Unità di Immunologia e Patologia Generale; Università di Pavia; Pavia, Italy.
authors.forename: Lucia Anna
authors.initials: LA
authors.lastname: Stivala
mesh_terms: D000595:Amino Acid Sequence; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D059748:Proteolysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays / Q000009:adverse effects*
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins
keywords: DDB2; Nucleotide Excision Repair; PCNA; UV damage; p21CDKN1A