Rad17 recruits the MRE11-RAD50-NBS1 complex to regulate the cellular response to DNA double-strand breaks

Abstract

The MRE11-RAD50-NBS1 (MRN) complex is essential for the detection of DNA double-strand breaks (DSBs) and initiation of DNA damage signaling. Here, we show that Rad17, a replication checkpoint protein, is required for the early recruitment of the MRN complex to the DSB site that is independent of MDC1 and contributes to ATM activation. Mechanistically, Rad17 is phosphorylated by ATM at a novel Thr622 site resulting in a direct interaction of Rad17 with NBS1, facilitating recruitment of the MRN complex and ATM to the DSB, thereby enhancing ATM signaling. Repetition of these events creates a positive feedback for Rad17-dependent activation of MRN/ATM signaling which appears to be a requisite for the activation of MDC1-dependent MRN complex recruitment. A point mutation of the Thr622 residue of Rad17 leads to a significant reduction in MRN/ATM signaling and homologous recombination repair, suggesting that Thr622 phosphorylation is important for regulation of the MRN/ATM signaling by Rad17. These findings suggest that Rad17 plays a critical role in the cellular response to DNA damage via regulation of the MRN/ATM pathway.

The MRE11 complex: An important source of stress relief

“…Following DNA damage, RAD17 localizes to lesions and facilitates the loading of the "9-1-1" sliding clamp complex (RAD9/HUS1/RAD1) that is known to play important roles in the activation of ATR and CHK1 [54]. The siRNA mediated depletion of RAD17 reduced ATM activation and impaired MRE11 complex retention at DSBs at times earlier than are affected by MDC1 depletion [55]. A direct interaction between the C-terminus of RAD17 and the NBS1-FHA domain was shown to be necessary for the retention of the MRE11 complex at breaks.…”

End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair

“…In other words, near DNA DSBs MRN is found in two different states, one dependent on phosphorylated H2AX via ATM, and the other independent of H2AX [114]. In addition, the replication checkpoint protein Rad17 is also phosphorylated by ATM at Thr622 site and this enables Rad17 to directly interact with Nbs1 leading to the recruitment of MRN complex to damaged sites [115].…”

Homologous DNA Repair:Safeguarding Genome Territories from Knives

“…DSBs are initially detected by chromatin associated fractions of MRN (MRE11/RAD50 (DNA recombinase)/ NBS1) complex 5,24,25 . This primary sensor sends signals and centrally governs the activation of ATM (AtaxiaTelangiectasia mutated) which subsequently phosophorylates a large cassette of downstream mediators including PAR (Poly-ADP Ribose) 24,25 , RAD17, MDC1, Ctlp, ChK1 (Checkpoint Kinase 1) ChK2 (Checkpoint Kinase 2), H2AX, BRCA1 (Breast Cancer susceptibity gene 1), CDK (Cyclin-Dependant Kinases), ATR (ATM-Rad3 related) and RPA (Replication Protein A), TOPB1 (DNA topoisomerase BPI) ( Figure 1).…”

“…This primary sensor sends signals and centrally governs the activation of ATM (AtaxiaTelangiectasia mutated) which subsequently phosophorylates a large cassette of downstream mediators including PAR (Poly-ADP Ribose) 24,25 , RAD17, MDC1, Ctlp, ChK1 (Checkpoint Kinase 1) ChK2 (Checkpoint Kinase 2), H2AX, BRCA1 (Breast Cancer susceptibity gene 1), CDK (Cyclin-Dependant Kinases), ATR (ATM-Rad3 related) and RPA (Replication Protein A), TOPB1 (DNA topoisomerase BPI) ( Figure 1). Subsequent retention and accumulation of MRN complex at damaged site is considered critical for HDR repair and is dependent on H2AX bound MDC1, ctlp, PAR and RAD17.…”

“…Subsequent retention and accumulation of MRN complex at damaged site is considered critical for HDR repair and is dependent on H2AX bound MDC1, ctlp, PAR and RAD17. Moreover, several evidences show that interaction with clamp loader RAD17 not only facilitates retention of MRN complex but also plays an important role in activation of ATR and CHK1 that along with CHK2 plays a role in cell cycle arrest in S and G2 phases 25 . In addition, ATR-CHK1 structure promote phosphorylation of RPA proteins independent of RAD17 for strand recovery when damaged DNA is resected 26 .…”