Torque Mediated Kinetics of CENP-A Nucleosomes Reveals Resilience to Tension

Abstract

In eukaryotic cells, DNA is wrapped around histone octamers, forming nucleosomes. In the centromere, the region of the chromosome that links sister chromatids, histone H3 is replaced by CENP-A (Centromere protein A). Since CENP-A chromatin is the point of contact between the microtubules/kinetochore complex and the rest of the chromosome, these regions endure very high forces. Whereas canonical nucleosomes unwrap at 3pN and disassemble at 15-20pN of force, the estimated forces applied by microtubili are much higher. To investigate how CENP-A nucleosomes respond to externally applied forces and torque, we studied how CENP-A chromatin responds to defined stretching forces and torque generated by magnetic tweezers. With this technique, a single DNA molecule containing a few (up to 10) nucleosomes is tethered between a glass surface and a magnetic bead. By applying stretching forces at constant negative and positive supercoiling, the forces needed for disassembly of canonical H3 and CENP-A nucleosomes can be obtained and compared. By measuring the DNA end-to-end length as a function of applied rotations before and after CENP-A nucleosomes are removed from the DNA, the linking number of the nucleosomes can be determined. Interestingly, while inducing supercoiling at constant low (<0.5pN) forces, unlike canonical nucleosomes, CENP-A nucleosomes appear to respond to the applied torque. By further analyzing the structural stability of CENP-A nucleosomes under stretching force and torque, we hope to unravel the mechanism by which CENP-A nucleosomes resists disassembly during mitosis in vivo.