PT  - JOURNAL ARTICLE
AU  - Rashmi Panigrahi
AU  - Ross Edwards
AU  - Md Touhidul (Apu) Islam
AU  - Jun Lu
AU  - Ayodeji Kulepa
AU  - Tae Hwan Kim
AU  - J. N. Mark Glover
TI  - Structural Insights into γH2Ax containing Nucleosomes
AID  - 10.1101/2023.04.30.538894
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.04.30.538894
4099  - http://biorxiv.org/content/early/2023/05/01/2023.04.30.538894.short
4100  - http://biorxiv.org/content/early/2023/05/01/2023.04.30.538894.full
AB  - 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 StatementThe authors have declared no competing interest.