Calling RNF168 to action.

Genomic stress leads to various forms of DNA damage, of which DNA double strand breaks (DSBs) are the most lethal. An army of signaling molecules is called to action as soon as these DNA breaks are detected. Various protein modifications, such as phosphorylation and ubiquitination, are an integral part of the reaction. While phosphorylation activates various proteins, ubiquitin (Ub) adducts typically act as docking sites for DNA repair factors. The response to DNA DSB starts with the protein kinase ATM phosphorylating various substrates including MDC1 and histone H2AX. This mediator protein, MDC1, then recognizes phosphorylated histone H2AX and amplifies the damage response. The E3 ligase, RNF8, recognizes and binds to phosphorylated MDC1. RNF8 then modifies an unknown protein to call a second ubiquitin ligase, RNF168, into action. It has been recognized that these two ubiquitin ligases are recruited sequentially but there is an unknown linker protein between them. These two ubiquitin ligases are crucial to the formation of DSB-associated ubiquitin conjugates and, as a result, there has been long standing interest in the field in identifying the link between the two factors. In this paper we identify lethal(3) malignant brain tumor like 2 (L3MBTL2) as the substrate of RNF8 (Nowsheen S, et al. Nat Cell Biol 20:455-464, 2018). We report that ATM-mediated phosphorylation of the polycomb group like protein L3MBTL2 and subsequent interaction with MDC1 brings it to the vicinity of the DNA lesion. RNF8 acts upon this phosphorylated L3MBTL2 and generates K63-linked polyubiquitin chains. This modified substrate is subsequently recognized by RNF168 and tethers the protein to the DNA lesion. RNF168 then ubiquitinates proteins such as histone H2A and H2AX to further amplify the damage response and recruit repair proteins such as BRCA1 and 53BP1 (Figure 1).