The talk will provide details on the design, characterization and applications of our avidity-based sensors to detect Ub–nucleosomes in mammalian cells. The main focus will be on our sensor to detect the DNA damage-associated H2AK13/15Ub. In the second part, I will present a protocol to assess the real-time recruitment of H2AK13/15Ub readers to DNA double-strand breaks. General details on sample preparation will be provided and more specific details can be provided upon request.

Our avidity-based sensors provide an alternative to conventional antibodies. These proteins can be genetically encoded, expressed in mammalian cells and used to assess the kinetics of formation and disappearance of specific histone ubiquitination signals in live cells in real-time (e.g., assess H2AK13/15Ub throughout the cell cycle). Moreover, these sensors can also detect histone ubiquitination in fixed samples. This approach is particularly attractive for histone PTMs for which there are no antibodies available.

The sensors do't utilize nano materials. For in vitro applications we express and purify the designed sensors from E. coli cells. For detection of histone ubiquitination in mammalian cells, the sensors are genetically encoded, fused to fluorescent tags (e.g., EGFP) and introduced into cells through transient or lentiviral transfection.

Nucleosome ubiquitination will have different functions depending on the histone and the lysine residue that are ubiquitinated. For example, H2AK13/15 ubiquitination is associated with the DNA damage repair response and it's downstream to H2AX phosphorylation by ATM/ATR kinases. More details will be provided in the introduction of this talk.