Abstract
MDC1 is a key mediator of DNA-damage signaling. When DNA double-strand breaks (DSB) occur, the histone variant H2AX on the nucleosome is phosphorylated on its C-terminus at residue Ser139 to form the γH2AX nucleosome. This phosphorylated form is specifically recognized by the tandem BRCT repeats of MDC1. The MDC1-bound nucleosome serves as a docking platform to promote the localization of other DNA repair factors. To further characterize the nucleosome-BRCT interaction, we developed a time efficient two-step modified native chemical ligation protocol to prepare phosphorylated nucleosomes. Our binding studies show that BRCT interacts with the nucleosome with a higher affinity than the phosphorylated peptide. Using cryogenic electron microscopy (cryo-EM), we obtained structures of the γH2AX nucleosome revealing the structural basis for nucleosome-nucleosome stacking promoted by interactions of the H4 N-terminal of one nucleosome with its stacked partner. In contrast, we show that binding of the MDC1 BRCT domain disrupts this stacking, suggesting that histone/DNA dynamics are integral to DNA damage signaling.
Competing Interest Statement
The authors have declared no competing interest.
