Regulation of the DNA Damage Response to DSBs by Post-Translational Modifications

Abstract

Damage to the genetic material can affect cellular function in many ways. Therefore, maintenance of the genetic integrity is of primary importance for all cells. Upon DNA damage, cells respond immediately with proliferation arrest and repair of the lesion or apoptosis. All these consequences require recognition of the lesion and transduction of the information to effector systems. The accomplishment of DNA repair, but also of cell cycle arrest and apoptosis furthermore requires protein-protein interactions and the formation of larger protein complexes. More recent research shows that the formation of many of these aggregates depends on post-translational modifications. In this article, we have summarized the different cellular events in response to a DNA double strand break, the most severe lesion of the DNA.

Keywords: DNA double strand breaks; cell cycle arrest; homologous recombination; non-homologous endjoining; post-translational modifications..

Fig. (1)

ATM and its target proteins. In the active state ATM phosphorylates target proteins that signal to cell cycle checkpoints, DNA repair proteins or to the cell death machinery. Phosphorylation of Brca1 or the activation of the MRN complex results in the initiation of DNA repair. Activation of cell cycle checkpoints that lead to cell cycle arrest are achieved by phosphorylation of the p53 tumor suppressor protein or the Chk2 kinase. In addition, by transcriptional induction of pro-apoptotic proteins like Bax and PUMA, p53 can also induce apoptosis.

Fig. (2)

ATM activation and downstream signaling. (A) ATM is present in the nucleus as an inactive dimer/multimer that is associated with PP2A, a phosphatase that controls ATM phosphorylation. Upon ionizing radiation (IR), ATM is activated by trans-autophosphorylation and dissociation of the dimer/multimer into monomers as well as by Tip60-mediated acetylation, which enables ATM-mediated phosphorylation of mediator and DNA repair proteins.

Fig. (3)

DNA lesion recognition and recruitment of repair factors. After introduction of a DNA double strand break (I), the DNA damage response is initiated by phosphorylation of H2AX (II), which is required for the recruitment of mediator proteins such as MDC1 or the MRN complex. MDC1 attaches to phosphorylated H2AX via its BRCT domain and stabilizes the MRN complex (III). Additionally it is responsible for binding and accumulation of ATM at the DSB, which creates a docking site for RNF8/Ubc13 that also associates with phosphorylated H2AX and decorates histone proteins with lysine 63-linked ubiquitin molecules (IV). RNF168 recognizes the initial ubiquitin chains generated by RNF8 and, in association with Ubc13, extends and propagates lysine 63-linked ubiquitination (V) that is required for the accumulation of additional repair factors like Bcra1 or 53BP1 (VI).

Fig. (4)

Mechanisms of DNA-repair by NHEJ and HR. Double strand breaks are repaired by two major pathways: non-homologous endjoining (A, NHEJ) and homologous recombination (B, HR). NHEJ: Loose DNA-ends are recognized by the Ku70/80 heterodimeric complex (A.I), which is needed for the recruitment of the additional repair factors DNA-PKcs, XRCC4, LigIV and XLF (A.II). Before broken DNA ends can be ligated, the ends are processed by Pol µ and λ, Artemis and Tdt (A.III). This step is followed by the ligation of the loose ends by LigIV and XRCC4 (A.IV). HR: HR is initiated by the MRN complex (B.I), followed by the recruitment of RPA that associates with single stranded DNA, and by Rad51/52 which are needed for filament formation and strand invasion (B.II). New DNA is synthesized while the homologous sister chromatid serves as a template (B.III). After branch migration, the holiday junction is resolved (B.IV).

Fig. (5)

Apoptosis signaling pathways. Apoptosis can be induced upon activation of death receptors (DR) at the membrane by binding of cognate ligands or after release of pro-apoptotic factors from mitochondria. Upon DR activation, the initiator-caspase-8 becomes activated, which activates a caspase-cascade, and cleaves the pro-apoptotic proteins Bid. The cleavage of Bid results in the formation of active tBid, which, in cooperation with Bax and Bak, forms pores in the outer mitochondrial membrane resulting in the release of cytochrome c, formation of the apoptosome and activation of caspase 9. Caspases 9 activates caspase 3 leading to the degradation of intracellular targets. The proapoptotic proteins Bax and Bak can be inhibited by Bcl-2 and Puma. Besides its nuclear function p53 can become monoubiquitinated in a Mdm2-dependent manner, which leads to the translocation of p53 to mitochondria where it participates in mitochondrial membrane permeabilization and cytochrome c release.