Molecular Biophysics of DNA repair nanomachines
Centro Nacional de Biotecnologia (CSIC)
C/Darwin 3, Campus de Cantoblanco 28049 Madrid, Spain
We investigated a protein nanomachine that we all need to repair inevitable damage to the DNA in our chromosomes, the Mre11 complex, an assembly of three proteins (Rad50, Mre11 and Nbs1). The Mre11 complex is essential for keeping our chromosomes together after DNA breaks, which occurs hundreds of times in all cells that are actively dividing. Un-repaired DNA breaks lead to cell death and incorrectly repaired breaks are a common cause of cancer. For instance, the Nbs1 component of this complex is defective in the genetic disease Nijmegen breakage syndrome, where patients have increased risk of developing cancer due to problems fixing chromosome breaks
TU Delft: Nynke Dekker and Cees Dekker
Erasmus MC Rotterdam: Martijn de Jager, Claire Wyman and Roland Kanaar
F. Moreno-Herrero et al. Nature 437 (7057), 440-443 (2005).
Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon DNA binding
We visualized and quantified the interaction between vaccinia topoisomerase IB (vTopIB) and DNA using the Atomic Force Microscopy. Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. Topoisomerases are essential for the survival of the cell and are a target for poisoning by anti-cancer drugs.
TU Delft: Laurent Holtzer, Daniel Koster, Cees Dekker and Nynke Dekker
Sloan-Kettering Institute: Stewart Shuman
F. Moreno-Herrero et al. Nucleic Acids Research 33(18), 5945-5953 (2005).
Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion
The mechanics of
DNA bending on intermediate length scales of 5100 nm plays a key
role in many cellular processes, and is also important in the fabrication
of artificial DNA structures, but prior experimental studies of DNA
mechanics have focused on longer length scales than these. We used high-resolution
atomic force microscopy (AFM) on individual DNA molecules to obtain
a direct measurement of the bending energy function appropriate for
scales down to 5 nm.
TU Delft: Thijn van der Heijden, Cees Dekker
Whitehead Institute: Paul wiggins
Stanford University: Andrew Spakowitz
Cal Tech: Rob Phillips
Northwestern University: John Widom
University of Pennsylvania: Philip Nelson
P.A. Wiggins et al. Nature Nanotechnology 1, 137-141 (2006).
High flexibility of DNA on short length scales probed by atomic force microscopy
Several bioinformatics studies have identified an unexpected but remarkably prevalent 10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. We investigated several hyperperiodic DNA segments from C.elegans using highresolution atomic force microscopy (AFM).
NOTE: This work was done in collaboration with Prof. Andrew Fire, who was awarded with the 2006 Nobel prize in Medicine.
TU Delft: Ralf Seidel and Nynke Dekker
Stanford University: Andrew Fire, Steven Johnson
F. Moreno-Herrero et al. Nucleic Acids Research 34(10), 3057-3066 (2006).
Structural analysis of hyperperiodic DNA from Caenorhabditis elegans
Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We measured the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy.
TU Delft: Thijn van der Heijden, Jeroen Abels, Cees Dekker and Nynke Dekker
J.A. Abels et al. Biophysical Journal 88(4), 2737-2744 (2005).
Single molecule measurements of the persistence length of double-stranded RNA