Chromatinization modulates topoisomerase II processivity

Abstract

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress. While cellular processes constantly create varying torsional stress, how this variation impacts type IIA topoisomerase function remains obscure. Using multiple single-molecule approaches, we examined the torsional dependence of eukaryotic topoisomerase II (topo II) activity on naked DNA and chromatin. We observed that topo II is ~50-fold more processive on buckled DNA than previously estimated. We further discovered that topo II relaxes supercoiled DNA prior to plectoneme formation, but with processivity reduced by ~100-fold. This relaxation decreases with diminishing torsion, consistent with topo II capturing transient DNA loops. Topo II retains high processivity on buckled chromatin (~10,000 turns) and becomes highly processive even on chromatin under low torsional stress (~1000 turns), consistent with chromatin’s predisposition to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function.

Optical Torque Calculations and Measurements for DNA Torsional Studies

“…Here, we measured DNA torsional response under a constant force (Fig. 6), which has clearly defined extension and torque characteristics (11,16,19,21,23,44,45): when turns are added to DNA, the DNA extension does not have significant change while torque in DNA increases linearly until the DNA buckles to extrude a plectoneme; after DNA buckling, DNA extension decreases while the torque remains constant. Since these features are intrinsic to DNA, they should be independent of the trap height and the cylinder displacement used in the measurements.…”

“…The quartz cylinders were fabricated by adapting protocols we previously established (11,19,23). We started the fabrication process from a 4-inch X-cut quartz wafer (Precision Micro-Optics).…”

“…The 6,481 bp DNA multi-labeled DNA was prepared via PCR from plasmid pMDW133 as described previously (20).…”

“…The angular optical trap (AOT) overcame this limitation, enabling simultaneous measurements of forces and torques of a biological molecule (9)(10)(11)(12). The AOT has been used to measure the torque generated by RNA polymerase (13,14), the torque required to destabilize a nucleosome (15), the torsional modulus of a single and braided chromatin fiber (16), and the force-torque relation of the DNA phase diagram (11,(17)(18)(19)(20)(21)(22)(23)(24).…”

“…An AOT requires a high numerical aperture (NA) objective to focus the trapping laser beam to a tight spot near the specimen plane. Thus far, DNA torque measurements and behaviors of DNA-based motor proteins have primarily relied on an oil-immersion objective (11,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). While an oil-immersion objective permits efficient trapping, it also inevitably introduces spherical aberrations at the trapping region due to the refractive index mismatch when the trapping beam passes through the interface between the glass coverslip and the aqueous solution of a sample chamber (Fig.…”