The role of 53BP1 in an ATM -dependent checkpoint pathway and the mechanism of its recruitment to DNA double strand breaks

53BP1 is a conserved nuclear protein that localizes rapidly at the sites of double strand breaks following damage. Using sIRNA technology we depleted 53BP1 from cells and monitored the effects on known markers of an active checkpoint response. We demonstrated that 53BP1 is required for cell cycle arrest at the G2/M transition and for the phosphorylation of at least a subset of ATM targets. Seeking to discover the mechanism by which 53BP1 is recruited to the sites of breaks, we solved the crystal structure of the domain necessary for 53BP1 to be recruited. It was found to consist of two tandem tudor folds with a conserved deep binding pocket formed at the interface between the folds. Mutation of the residues that make up this binding pocket abolish the ability of 53BP1 to localize at the sites of double strand breaks. In vitro binding experiments showed that this tandem tudor domain bound histone H3 that was methylated on lysine 79 and in vivo crosslinking experiments confirmed this finding. Moreover, suppression of DOT1L, the methyltransferase responsible for modifying lysine 79 of histone H3, also inhibited the recruitment of 53BP1 to double strand breaks. As the levels of lysine 79 remained unaltered after DNA damage, we conclude that double strand breaks causes a change in chromatin structure that exposes the 53BP1 binding site