Single-molecule techniques are powerful tools in studying functions of biological entities by investigating individual molecules. In this talk, I would like to discuss how single-molecule DNA flow-stretching assay can help elucidating underlying working mechanisms of DNA-binding proteins using ParB protein as an example.
ParB proteins play a crucial role in low-copy-number plasmid and chromosome partitioning and segregation in bacteria. They bind to specific DNA sequences called parS with a high affinity, but ParB proteins associate with 10-20 kb region encompassing parS, a phenomenon termed spreading. Recent paradigm-shifting discoveries revealed that ParB protein is a novel enzyme that utilizes cytidine triphosphate (CTP) to modulate its functions. We employed single-molecule techniques to elucidate roles of CTP and found that CTP prevents random recruitments of BsParB proteins. If time permits, I will discuss our surprising finding that a small KCK-tag that is commonly used in fluorescent dye labeling on a protein leads to unexpected artifacts.
