Single-molecule Studies of Telomere Maintenance

Telomeres play important roles in maintaining the stability of linear chromosomes. The telomeric structure allows a cell to distinguish between natural chromosome ends and double-stranded DNA breaks. Telomere dysfunction and associated chromosomal abnormalities have been strongly associated with age-associated degenerative diseases and cancer. Telomere maintenance involves dynamic actions of multiple proteins on a long complex DNA structure. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. Ongoing research in our laboratory involves the application of a broad range of biochemical and biophysical assays, including single-molecule atomic force microscopy (AFM) and fluorescence imaging, to study the conformation and dynamics of proteins involved in telomere maintenance.

AFM and DREEM

AFM generates an image of a surface by scanning with a sharp sensor tip attached to a cantilever. The most commonly applied AFM imaging modes are contact mode, intermittent contact (or oscillating) mode, and noncontact mode. Many protein-protein and protein-DNA complexes have been imaged in air and under solution at nanometer resolutions, establishing AFM as a versatile imaging tool for studying these biological systems. In addition, we apply Dual-Resonance-frequency-Enhanced EFM (DREEM) developed by the Erie lab at UNC Chapel Hill to resovle ds and ssDNA within protein-DNA complexes.

DNA tightrope assay

Dynamic movements on DNA, such as 1-dimensional (1-D) sliding (translocation while maintaining continuous DNA contact), jumping and hopping (microscopic dissociation and rebinding events), are essential for a protein to achieve its function inside cells where nonspecific DNA is in vast excess and bound by histones and other proteins. To track dynamic motion of proteins on DNA, we (in collabration with Neil Kad), developed a holistic approach using quantum dot labeled proteins and a DNA tightrope assay to overcome the key imaging limitations:

Microscopes



The MFP-3D-BIO Atomic Force Microscope (AFM) from Asylum Research is a high performance AFM specifically designed for biological applications. This microscope is equipped with a BioHeater, an Environmental Controller, and an iDrive Kit for visualizing protein-DNA samples in solution. Scan Size: X&Y axes: 90um range in closed loop; Resolution: 0.25 nm. It can carry "autoscan" to automatically scan a 90 um by 90 um area. A Stanford Research SR844RF lockin amplifier and 3.1MHz synthesized function generator are used for the new DREEM imaging technique.




This Nikon microscope is equipped with an encoded motorized stage, perfect focus system (PFS), a Ti-TIRF E motorized illuminator unit,
a 488 nm solid state laser, a 100X TIRF objective, a Dual View device, and an electron multiplied (EM) CCD camera (iXon DU897, Andor Technology).
The excitation beam is reflected into the objective through a TIRF filter set containing zt488rdc and ET500LP filters. For simultaneous imaging of green (565 nm)
and red (655 nm) Qdots, a dual view simultaneous imaging system was used in combination with a T605LPXR dichroic beam splitter and a bandpass filter ET655/40m (Chroma).