Research Topics, Projects & Instrumentation

Dynamic Force Spectroscopy on 30 nm fibers

Arrays of of nucleosomes fold into a compact 30 nm diameter fiber under phsiological conditons . This dense structure ensures a high degree of DNA compaction and plays a key role in the regulation of all biological functions involving genomic DNA like replication, transcription and repair. Although a wealth of physical models for chromatin structure and mechanics has been proposed in literature, experimental validation of these models has proven difficult. The anticipated highly dynamic state of chromatin, that is required for biological functionality, further complicates this issue. As a result the geometry and dynamics of higher-order structures – beyond the nucleosome – has been debated for 30 years.

 

In our group we have developed Magnetic Tweezers based dynamic force spectroscopy, uniquely capable of probing (transient) mechanical properties of chromatin fibers at forces between 10fN and 10pN. The accessible force range is significantly below that of competing techniques like Optical Tweezers and Atomic Force Microscopy. Whereas others were forced to focus in similar experiments on the high force regime (> 5pN), and thereby disrupting all chromatin structure except for the final wrap of DNA around the histone octamer, we were recently able to probe the compliance of the intact 30 nm fiber.

 

DNA breathing in single nucleosomes

DNA wrapping around a core of histone proteins significantly hinders interaction with DNA processing enzymes. The nucleosome however, has been proposed to relieve this steric constraint by its remarkable ability to transiently release part of its DNA, a process referred to a DNA breathing. It has been shown that DNA that is located near the center diad of the nucleosome is significantly less accessible than DNA near the entry of a nucleosome. The dynamics of this process has only recently been addressed, primarily by using techniques based on Fluorescence (or Förster) Resonance Energy Transfer (FRET). FRET is a process in which the energy of an excited donor fluorophore is transferred non-radiatively to an acceptor molecule. The efficiency of energy transfer is strongly dependent on the distance between the two fluorophores, with a typical decay length of 5 nm. Coincidentally, this is also the radius of the nucleosome, making FRET a well suited ruler to measure (changes in) distances between two points on the nucleosome.

 

By reconstitution of nucleosomes with DNA molecules that contain fluorophores 75 bp apart we have an system of which the FRET efficiency is extremely sensitive for DNA reorganization. The breathing of DNA in a nucleosome is typically driven by thermal fluctuations, resulting in unsynchronized, stochastical changes in FRET efficiency. In order to resolve the dynamics of DNA breathing we used Total Internal Reflection (TIRF) based wide field microscopy. For the first time, we were able to unambiguously attribute fluctuations in FRET efficiency to DNA breathing, paving the path for studying more intricate mechanisms of DNA dynamics.

 

Nucleosome translocation by ATP- dependent remodelers

ATP-dependent remodelers continuously modify the structure of chromatin to accommodate genome transactions. One of these remodelers is the Remodeling Structure of Chromatin (RSC) complex. RSC, like other ATP dependent chromatin remodelers, contains a motor subunit from the DEAD-box family, which tracks over DNA, and moves nucleosomes on the way. Revealing the detailed mechanism of this process is a challenging task, for which we have employed Atomic Force Microscopy (AFM).

 

With AFM we visualized individual RSC complexes on single nucleosome substrates. Multiple lobes of the RSC complex were resolved in close agreement with previously reported EM tomographs. Upon incubation with ATP, nucleosomes were displaced from the central positioning element towards the ends of the DNA. From complexes trapped during remodeling and the distribution of nucleosomes before and after remodeling the mechanism of remodeling could be deduced, in which RSC binds to a DNA end, translocates along DNA and drags the nucleosome with it. The mechanism was quantitatively tested using Monte Carlo simulations, from which the processivity of DNA translocation by RSC can be obtained.

 

Projects

  • DNA in action: Physics of the Genome. 2007 Programmaruimte FOM
  • Putting one and one together: the physics of more than one nucleosome. 2007 Projectruimte FOM
  • Resolving Structural and Mechanical Properties of Chromatin Fibers. 2007 LION
  • Single-molecule Kinetics of Chromatin Organization. 2004 BUW, FOM
  • Chromatin Higher Order Dynamics: A single molecule approach. 2004 EuroDYNA, ESF
  • Single-Molecule Dynamics of Chromatin Remodelling; Getting Around Nucleosomes. 2003 VIDI, NWO natuurkunde

 

Instrumentation

  • Magnetic Tweezers
  • Optical Tweezers
  • TIRF Fluoresence Microscopy
  • Atomic Force Microscopy
  • Fluoresence Correlation Spectroscopy


Next to these instruments that were developed in the lab we have a range of standard optical and biochemical equipment available at the Institute of Physics or in service facilities at the Faculty of Sciences. We welcome visitors to our laboratory for joined research projects or initial proof-of-principle experiments which might lead to a joined proposal. If you have interest in a trial experiments you should contact John van Noort.