Inhibition of DNA damage repair by artificial activation of PARP with siDNA

• Published in: Nucleic acids research 2013-08, Vol.41 (15), p.7344-7355
• Main Authors: Croset, Amelie, Cordelières, Fabrice P, Berthault, Nathalie, Buhler, Cyril, Sun, Jian-Sheng, Quanz, Maria, Dutreix, Marie
• Format: Article
• Language: English
• Subjects: Base Sequence; Benzimidazoles - pharmacology; BRCA2 Protein - genetics; BRCA2 Protein - metabolism; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; DNA-Activated Protein Kinase - genetics; DNA-Activated Protein Kinase - metabolism; Enzyme Activation; Genome Integrity, Repair and; Genome, Human; HeLa Cells; Humans; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases - genetics; Poly(ADP-ribose) Polymerases - metabolism; Signal Transduction
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkt522

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.