Abstract
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.
Highlights
To overcome DNA damage, cells have evolved mechanisms to detect DNA lesions, signal their presence and promote their repair [1,2,3]
The main pathway for the repair of DNA double-strand breaks (DSB) is non-homologous end-joining (NHEJ) that depends on DNAdependent protein kinase (DNA-PK); when NHEJ is impaired, an alternative or back-up NHEJ (B-NHEJ) pathway dependent of poly [Adenosine Diphosphate (ADP)-ribose] polymerase (PARP) operates [34]
Accessory proteins control a hierarchy in which DNA-PKdependent regular NHEJ repair is privileged over PARPdependent B-NHEJ [4,35]
Summary
To overcome DNA damage, cells have evolved mechanisms to detect DNA lesions, signal their presence and promote their repair [1,2,3]. Responses to different classes of DNA lesions differ, most occur via signal transduction cascades involving post-translational modifications, such as ubiquitination, phosphorylation, acetylation and poly (ADP-ribosy)lation (PAR called PARylation). Key regulators within these signaling cascades, such as the phosphatidylinositol 3-kinase-related kinases (PI3K) Ataxia Telangiectasia Mutated (ATM), Ataxia Telangiectasia and Rad3-related (ATR) or DNAdependent protein kinase (DNA-PK) and the poly [Adenosine Diphosphate (ADP)-ribose] polymerase (PARP), are activated via direct or indirect interaction with double-strand breaks (DSB) and single-strand breaks (SSB) [4,5,6]. Cells have two major pathways to repair DSB: homologous recombination (HR) and non-homologous end-joining (NHEJ) [7,8].
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