Assembly of checkpoint and repair machineries at DNA damage sites

Abstract

The remarkably coordinated nature of the DNA damage response pathway relies on numerous mechanisms that facilitate the assembly of checkpoint and repair factors at DNA breaks. Post-translational modifications on and around chromatin have critical roles in allowing the timely and sequential assembly of DNA damage responsive elements at the vicinity of DNA breaks. Notably, recent advances in forward genetics and proteomics-based approaches have enabled the identification of novel components within the DNA damage response pathway, providing a more comprehensive picture of the molecular network that assists in the detection and propagation of DNA damage signals.

Figure 1. MDC1 is the molecular platform for the assembly of checkpoint and repair proteins at the vicinity of DNA damage sites

Schematic illustration of MDC1 domain organization and their respective roles in the assembly of various components in the DNA damage response pathway. i) Upon DNA damage, the histone variant H2AX (orange) surrounding DNA damage sites is phosphorylated by the ATM, DNA-PK and ATR kinases, which allows the direct engagement of MDC through its phospho-protein binding BRCT domain (black). ii) At damaged chromatin, the MDC1 FHA domain (blue) recruits phosphorylated ATM (gray) molecules and allows amplification of ATM signaling. iii) The MDC1 tandem SDTD repeats (aqua) are phosphorylated by casein kinase 2, which allows accumulation of the MRN complex through its interaction with NBS1. iv) Phosphorylation of the tandem TQXF repeats (lime green) on MDC1 mediates its interaction with RNF8 through its FHA domain, which initiates the ubiquitin-dependent signaling pathway involving RNF8, RNF168 and UBC13.

Figure 2. Regulatory ubiquitylation-dependent signaling cascade involving RNF8 and RNF168

Pictorial representation of the RNF8 and RNF168-mediated substrate ubiquitylation at DSBs to facilitate accumulation of various DNA damage checkpoint and repair proteins. DNA damage triggers the phosphorylation of the histone variant H2AX (orange) at the vicinity of DNA breaks, which in turn directly recruits MDC1 through its phospho-protein binding BRCT domain. Phosphorylated MDC1 then allows accumulation of the E3 ubiquitin ligase RNF8 (dark blue), which in concert with the E2 ubiquitin conjugating enzyme UBC13 (blue), mediates H2A-type histone ubiquitylation. RNF168 (pink) recognizes the RNF8-mediated ubiquitylated histone, and with UBC13, further amplifies the local ubiquitin environment, which is required for focal accumulation of downstream checkpoint and repair factors, including RAP80 (blue), 53BP1 (green), RAD18 (orange) and PTIP (dark blue). Whether histone H2A (dark blue) is poly-ubiquitylated by K63-linked ubiquitin chains remains to be determined. In addition, exactly how RNF168–UBC13-dependent substrate ubiquitylation mediates the recruitment of the downstream DNA damage responsive elements remains unclear. Ubiquitin: ub (light blue).