Molecular mechanism of polyamines on DNA dynamics during DNA repair

PROJECT SUMMARY Polyamines (PAs) are positively charged, branched small molecules that are ubiquitous throughout the cell, heavily concentrated within the nucleus, and are frequently found bound to negatively charged nucleic acids and proteins. PAs play regulatory roles within the cell due to their ability to bridge molecules intra- and intermolecularly, leading to the condensation and compaction of DNA, nucleosomes, and chromatin. Despite their small size, PAs participate in genomic spatial and topological organization, playing roles in genome integrity and gene expression. With Parkinson’s disease, polyamine homeostasis is perturbed. In addition, DNA damage levels are elevated and are not efficiently repaired, eventually leading to neurodegeneration and cell death. This study will investigate and determine the molecular mechanism of polyamines on DNA dynamics during DNA repair by homologous recombination (HR). It will utilize in vitro single-molecule and bulk solution assays to assess the extent to which PAs impact the flexibility and dynamics of single-stranded, double-stranded, and non- canonical DNA structures to understand how PAs can impact the dynamics of their formation and accumulation (Aim 1). The in vitro assays will be extended by adding RAD51 to understand the role of PAs in protein-DNA interactions and their impact on RAD51-mediated strand exchange and repair (Aim 2). This will be followed by live cell fluorescence microscopy and chromatin immunoprecipitation sequencing of RAD51-mediated repair in a PA-dependent manner to visualize and quantify the role of PAs in DNA damage repair (Aim 3). The combination of Sua Myong and Taekjip Ha will foster a strong training environment. The Myong lab is one of the leaders in studying non-canonical nucleic acid conformations and protein-nucleic acid interactions leveraging novel single-molecule and cellular approaches. The Ha lab is also a leader in visualizing and quantifying genome maintenance and mechanics by developing and using fluorescence imaging approaches in cellulo combined with CRISPR technology. The mentorship team of both Sua Myong and Taekjip Ha will cover the biophysical and genome topics necessary to build a strong academic career in tackling health questions through a basic science research lens.