Enhanced Electrostatic Force Microscopy Imaging Reveals Mechanism of TRF2 Mediated DNA Compaction

Abstract

T-loop formation at telomeres is proposed to play an important role in telomere protection through the sequestration of the 3’ single-stranded overhang. Previous studies indicated that shelterin protein TRF2 modulates telomere structure by promoting DNA compaction and T-loop formation. To further understand the mechanism underlying TRF2-mediated DNA compaction, we applied Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM), a recently developed technique capable of high-resolution imaging of weak electrostatic potentials. DREEM images of nucleosomes clearly reveal DNA strands wrapped around histone proteins in nucleosomal arrays. In contrast, DREEM imaging shows DNA compacted inside TRF2 complexes through a 3-dimensional stacking of TRF2 dimers mediated by collective actions of multiple copies of TRF2 proteins. TRF2-mediated DNA compaction leads to electric potential gradients across the complexes. Surprisingly, while DNA wrapped around histones displays similar electrostatic potential signals compared to bare DNA, TRF2 DNA compaction leads to significant differences in the electrical potential signals inside multi-oligomeric TRF2 complexes compared to protein or DNA alone. These results clearly demonstrate the electrostatic changes in TRF2-DNA complexes upon oligomerizaton of proteins and DNA compaction, and underscore the importance of developing new electrostatic force microscopy imaging techniques for studying biological systems.