Measurements of Force-Driven Changes in Bound Protein Numbers on a Single DNA

Abstract

DNA compaction and chromosome organization involve the dynamic interaction of long DNA molecules and many copies of various proteins. Determining numbers of proteins bound to large DNAs is important to understand their chromosomal functions and protein numbers may be affected not only by chemical factors, but also by physical factors such as mechanical forces generated in DNA, e.g., by transcription or replication (1). We performed single-DNA stretching experiments with bacterial nucleoid proteins HU (2) and Fis, where we verified that the force-extension measurements were in thermodynamic (chemical-mechanical) equilibrium. Given thermal equilibrium of protein binding, we could use a thermodynamic Maxwell relation to deduce the change of protein number on a single stretched DNA due to varied applied force. For the binding of both HU and Fis under conditions where they compact DNA, the numbers of bound proteins decreased as force was increased from 0.03 to 12 pN. This effect saturated with force for HU, but did not for Fis, reflecting the tighter binding of the latter to DNA. The experimental results agree well with expectations of binding numbers based on electrophoretic mobility shift assay data, and the HU data agree well with results from a simple statistical-mechanical model of DNA-bending proteins. This thermodynamic approach may be applied to measure force-driven changes in numbers of a wide variety of molecules bound to DNA or to other polymers; in the case of proteins binding to DNA, force-dependent binding suggests mechano-chemical mechanisms for gene regulation. 1. C. Bustamante, Z. Bryant, S. B. Smith, Nature, (2003). 2. B. Xiao, R. C. Johnson, J. F. Marko, Nucleic Acids Res, (2010).