DNA double-strand break signaling and human disorders

Abstract

DNA double-strand breaks are among the most serious types of DNA damage and their signaling and repair is critical for all cells and organisms. The repair of both induced and programmed DNA breaks is fundamental as demonstrated by the many human syndromes, neurodegenerative diseases, immunodeficiency and cancer associated with defective repair of these DNA lesions. Homologous recombination and non-homologous end-joining pathways are the two major DNA repair pathways responsible for mediating the repair of DNA double-strand breaks. The signaling of DNA double-strand breaks is critical for cells to orchestrate the repair pathways and maintain genomic integrity. This signaling network is highly regulated and involves a growing number of proteins and elaborated posttranslational modifications including phosphorylation and ubiquitylation. Here, we highlight the recent progress in the signaling of DNA double-strand breaks, the major proteins and posttranslational modifications involved and the diseases and syndromes associated with impaired signaling of these breaks.

Figure 1

Mammalian DNA damage repair pathways and checkpoints. Various exogenous and endogenous sources can generate damaged DNA. In response to DNA damage cells activate the appropriate DNA damage repair and checkpoint pathways or apoptosis. Lesions of single or double-stranded DNA lead to the activation of cell cycle checkpoints and a number of DNA damage repair pathways including MMR (mismatch repair), BER (base excision repair), NER (nucleotide excision repair), TLS (translesion DNA synthesis), HR (homologous recombination) and NHEJ (non-homologous end joining). Impaired repair of damaged DNA can lead to accumulation of, mutations and genomic instability. In addition, defective repair of damaged DNA can lead to aging as well as predisposition for various genetic diseases including cancer and immunodeficiency.

Figure 2

Schematic representation of the DNA damage-signaling that leads to activation of cell cycle checkpoints or apoptosis. (A) Examples of proteins involved in the different steps of DNA damage signaling are shown. DNA lesions are recognized by sensors (e.g ATM), and mediators (e.g 53BP1) serve to amplify the signaling of DNA damage. Next, proteins including CHK2 serve to transduce the DNA damage signals. Finally, effectors (e.g p53) are required to trigger the appropriate DNA damage cellular responses that include apoptosis, senescence or cell cycle arrest or delay that allow cells to repair their damaged DNA. (B) In response to DSBs and/or DNA replication failure, activated ATM and/or ATR phosphorylate the CHK2 and CHK1. Activated ATM, ATR and CHK2 also phosphorylate p53, thus increasing its stability and activation. Activated p53 transactivates the p21 that inhibits the cyclin-dependent-kinases and delays the G1/S transition. In the case the damage DNA is beyond repair, p53 can promote apoptosis of the damaged cells through the transactivation of its transcriptional targets including the Bax, Puma and Noxa. CHK1 is essential for S and G2/M checkpoints activation. CDC25C inactivation and WEE1 activation through their phosphorylation by CHK1 result in the inhibition of CDC2/cyclin B activity and G2/M arrest. CDC25C dephosphorylates CDC2 leading to its activation [177]. In response to DNA damage, CHK1 phosphorylates CDC25C allowing its interaction with 14-3-3 and inhibition of its phosphatase activity [178,179]. CHK1 also phosphorylates WEE1, affecting its distribution through interaction with 14-3-3. Finally, CHK1 also phosphorylates CDC25A leading to CDK2 inactivation and delayed intra-S phase [180].

Figure 3

Multistep signaling of DNA double-stranded breaks. ATM and ATR, through the phosphorylation of their downstream substrates, play central roles in DSB signaling and the activation of cell cycle checkpoints. (A) Mre11/Rad50/NBS1 (MRN)-complex mainly recognizes DSBs and undergo ATM activation. Activated ATM phophorylates multiple substrates, including H2A.X and MDC1. MDC1 recognizes γ-H2A.X and bind to it. Then RNF8 recognizes the thyreonyl-phosphorylated MDC1 via its FHA domain and is recruited to DSB sites where it ubiquitylates the histone H2A. Subsequently, RNF168 is recruited to the ubiquitylated H2A, further ubiquitylating it with Ubc13 and likely leading to the modification of the chromatin structure. Through its tutor domain, 53BP1 is recruited to histone H4 di-methylated on lysine 20, while RAP80 and its protein complex are recruited to the polyubiquitylated H2A. (B) In the S and G2 phase, DSBs are recognized by the MRN-complex and CtIP is recruited to DSBs. CtIP is phosphorylated by CDK and ATM, and is ubiquitylated by BRCA1 in response to DNA damage. Collaboration of MRN and CtIP results in DSB end resection, and subsequently DNA2/BLM-complex and Exo1 promote further the resection of DSB ends leading to generation of single strand DNA (ssDNA). RPA binds to the ssDNA and induces ATR activation and HR repair [181-184]. (C) Replication fork stalling. The damaged single-stranded DNA is coated by RPA and this structure plays crucial roles in the recruitment of the helicase HARP and the ATR-ATRIP complex to sites of DNA damage[185-187]. P: phosphorylation, Ub: ubiquitin, S: SUMOylation, ATRIP: ATR interacting protein, RPA: Replication protein A. HARP: HepA-related protein.