RAP80 and RNF8, key players in the recruitment of repair proteins to DNA damage sites

Abstract

Chromosomal double-strand breaks (DSBs) in eukaryotes provoke a rapid, extensive modification in chromatin flanking the breaks. The DNA damage response (DDR) coordinates activation of cell cycle checkpoints, apoptosis, and DNA repair networks, to ensure accurate repair and genomic integrity. The checkpoint kinase ATM plays a critical role in the initiation of DDR in response to DSBs. The early ATM-mediated phosphorylation of the histone variant H2AX proteins near DSBs leads to the subsequent binding of MDC1, which functions as a scaffold for the recruitment and assembly of many DDR mediators and effectors, including BRCA1. Recent studies have provided new insights into the mechanism by which BRCA1 and associated proteins are recruited to DNA damage foci and revealed key roles for the receptor-associated protein 80 (RAP80) and the E3 ligase RNF8 in this process. RAP80 is an ubiquitin-interaction motif (UIM) containing protein that is associated with a BRCA1/BARD1 complex through its interaction with CCDC98 (Abraxas). The UIMs of RAP80 are critical for targeting this protein complex to DSB sites. Additional studies revealed that after binding gamma-H2AX, ATM-phosphorylated MDC1 is recognized by the FHA domain of RNF8, which subsequently binds the E2 conjugating enzyme UBC13. This complex catalyzes K63-linked polyubiquitination of histones H2A and gamma-H2AX, which are then recognized by the UIMs of RAP80, thereby facilitating the recruitment of the BRCA1/BARD1/CCDC98/RAP80 protein complex to DSB sites. Depletion of RAP80 or RNF8 impairs the translocation of BRCA1 to DNA damage sites and results in defective cell cycle checkpoint control and DSB repair. In this review, we discuss this cascade of protein phosphorylation and ubiquitination and the role it plays in the control of cellular responses to genotoxic stress by regulating the interactions, localization, and function of DDR proteins.

Fig. 1

Schematic illustration of the hierarchical functions of ATM, H2AX, MDC1, RNF8 UBC13, CCDC98/Abraxas, and RAP80 in the recruitment of BRCA1-BARD1 protein complexes to DSB sites. (A) Induction of DSBs by ionizing irradiation (IR) results in the recruitment of ATM by the MRN sensor complex and ATM activation. (B) ATM then catalyzes the phosphorylation of H2AX, which subsequently binds MDC1 through its BRCT domain. RAP80 is part of a BRCA1-BARD1-CCDC98 protein complex through its interaction with CCDC98/Abraxas and an early target for ATM-mediated phosphorylation. (C) After binding γ-H2AX, ATM-phosphorylated MDC1 functions as a scaffold for the recruitment and assembly of DDR mediators and effectors. Phosphorylated MDC1 is recognized by the FHA domain of RNF8, which subsequently binds the E2 conjugating enzyme UBC13. (D) This complex then catalyzes K63-linked polyubiquitination of histones H2A and γH2AX. These polyubiquitin chains are recognized by the UIMs of RAP80, thereby facilitating the recruitment of the BRCA1/BARD1/CCDC98/RAP80 protein complex to DSB sites.

Fig. 1

Schematic illustration of the hierarchical functions of ATM, H2AX, MDC1, RNF8 UBC13, CCDC98/Abraxas, and RAP80 in the recruitment of BRCA1-BARD1 protein complexes to DSB sites. (A) Induction of DSBs by ionizing irradiation (IR) results in the recruitment of ATM by the MRN sensor complex and ATM activation. (B) ATM then catalyzes the phosphorylation of H2AX, which subsequently binds MDC1 through its BRCT domain. RAP80 is part of a BRCA1-BARD1-CCDC98 protein complex through its interaction with CCDC98/Abraxas and an early target for ATM-mediated phosphorylation. (C) After binding γ-H2AX, ATM-phosphorylated MDC1 functions as a scaffold for the recruitment and assembly of DDR mediators and effectors. Phosphorylated MDC1 is recognized by the FHA domain of RNF8, which subsequently binds the E2 conjugating enzyme UBC13. (D) This complex then catalyzes K63-linked polyubiquitination of histones H2A and γH2AX. These polyubiquitin chains are recognized by the UIMs of RAP80, thereby facilitating the recruitment of the BRCA1/BARD1/CCDC98/RAP80 protein complex to DSB sites.