Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Linda Yarfi - Kean University
Co-Author(s): Carolina Dos Santos Passos, Colorado State University, Fort Collins, CO ; Yun Choi, Colorado State University, Fort Collins, CO ; Tingting Yao, Colorado State University, Fort Collins, CO; Robert Cohen, Colorado State University, Fort Collins, CO
Histone post-translational modifications (PTMs) have a role in regulating processes as gene transcription, DNA replication, and DNA damage and repair (DDR). Upon DNA double-strand breaks (DSBs), H2AK13/15 is ubiquitinated by the E3 ligase RNF168, and the resulting H2AK13/15-Ub nucleosomes contribute to the recruitment of repair proteins to the break sites. Yao’s & Cohen’s research groups have been working in the design of protein sensors targeting H2AK13/15-Ub nucleosomes as tools to understand the events triggered by these PTMs upon DNA damage. Among these sensors, the prototype Reader 1.0 has shown to co-localize with the repair protein 53BP1 in DSB sites. However, Reader 1.0 was not able to differentiate between H2AK15-Ub and H2BK120-Ub in vitro, which is in agreement with the spatial proximity of these two ubiquitination sites in the nucleosome. Here, we aim to investigate five variations of Reader 1.0 (Fig. 1A) obtained by increasing the length of the peptide linker and changing its flexibility. Our goals are to improve the selectivity towards H2AK13-Ub while keeping the sensors’ ability to co-localize with ubiquitinated nucleosomes in mammalian cells. We decided to look for sensors targeting H2AK13-Ub nucleosomes considering that this ubiquitination site is two residues away from H2AK15 and that this distance could be exploited to improve sensor’s selectivity and to decrease its binding affinity for H2BK120-Ub. Taking these goals into account, our approach will be first to verify the sensors’ co-localization with damaged DNA in mammalian cells, and further to investigate sensors’ selectivity towards H2AK13-Ub nucleosomes in vitro.Not Submitted
Funder Acknowledgement(s): National Science Foundation
Faculty Advisor: Tingting Yao, PhD, Tingting.Yao@colostate.edu
Role: I participated in every part if this project except the design for the prototype peptide (Reader 1.0). This part was completed by the research assistant in the lab (Yun Choi). I participated in the cloning processes to generate our protein, the purification processes, and the analysis process.