Molecular Biophysics of DNA repair nanomachines
Centro Nacional de Biotecnologia (CSIC)
C/Darwin 3, Campus de Cantoblanco 28049 Madrid, Spain
Our group started in 2009 at the National Center of Biotechnology at
Madrid with the aim of understand how protein machines involved in DNA
repair and chromosome organisation work at the single molecule level. To do that, we use single-molecule
techniques such as Atomic Force Microscopy, Magnetic Tweezers and Optical tweezers.
Up to date we have two functional AFMs from Nanotec Electronica and two custom-made Magnetic Tweezers setups.
We have also built an Optical Tweezers setup and have access to standard biochemical techniques for
nucleic acids manipulation and characterization.
Here are two pictures of one of our MTs and AFMs (enclosed in a sound-proof box).
This is one of the many projects started with the group of Dr. Mark Dillingham from
University of Bristol in the context of our Starting Grant ERC Project.
Recombinational repair of DNA breaks requires processing of a DNA end to a 3-ssDNA overhang. In B.subtilis, this task is done by the helicase-nuclease AddAB which generates ssDNA overhangs terminated in a recombination hotspot CHI sequence. This is a substrate for the formation of a RecA nucleoprotein filament that searches for a homologous donor molecule and catalyses DNA strand exchange to promote repair. In this project, we have used AFM and Magnetic Tweezers to visualize and characterize the products of reactions including AddAB and double-stranded DNA molecules, and to monitor the real time dynamics of DNA translocation and unwinding.
Our work and that of others have provided an exquisite understanding of the end-processing step for DNA repair by homologous recombination in Bacteria.
University of Bristol: Mark Dillingham Group
Yeeles et al. Molecular Cell 42, 806-816 (2011).
Recombination Hotspots and Single-Stranded DNA Binding Proteins Couple DNA Translocation to DNA Unwinding by the AddAB Helicase-Nuclease
Carrasco et al. PNAS 110 (28), E2562-2571 (2013).
On the mechanism of recombination hotspot scanning during double-stranded DNA break resection
Carrasco et al. DNA Repair 20, 119-129 (2014) (cover article).
Single molecule approaches to monitor the recognition and resection of double-stranded DNA breaks during homologous recombination
Gilhooly et al. Nucleic Acids Research 44(6), 2727-2741 Published Online Jan 13 (2016).
Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination
This is the main current project supported by an ERC Consolidator Grant 2015. We aim to understand the mechanism of
action of SMC complexes, including understanding the role of SMC loaders and SMC accessory subunits, and how these
proteins are regulated by ATP binding and hydrolysis for chromosome organisation.
We have done preliminary work in the characterisation of the SMC complex with AFM. Using an innovative approach that uses DNA as a fiducial marker to quantify volumes of proteins with high precision, we determined the oligomeric states and architecture of the Bacillus subtilis SMC complex. AFM results were used to color code the different protein components of the SMC complex: monomers of ScpA (pink); monomers and dimers of ScpB (green) ; ScpA-ScpB complexes (yellow); and SMC proteins (blue). The fiducial DNA molecule used in the study appears in white at the bottom part of the picture.
University of Bristol: Mark Dillingham Group
M.E. Fuentes-Perez et al.Biophysical Journal 102, 839-848 (2012).
Using DNA as a fiducial marker to study SMC complex interactions with the Atomic Force Micrsocope
M.E. Fuentes-Perez et al.METHODS 60, 113-121 (2013).
AFM volumetric methods for the characterization of proteins and nucleic acids
Taylor*, Pastrana* et al. Nucleic Acids Research 43(2), 719-731 (2015).
Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation
We have investigated the mechanical properties of dsDNA and dsRNA using AFM, Magnetic Tweezers, and Optical Tweezers. GC-rich DNA shows in circular-dicroism experiments a signal compatible with A-form DNA. We investigated the mechanical properties and stability conditions of GC-rich DNA and compared these with a control DNA of equal GC/AT content under a variety of buffers and a wide range of forces. To do this study we used Magnetic Tweezers and AFM.
IMDEA Nanociencia: B. Ibarra´s Group, R. Arias-Gonzalez´s Group
UAM. Science Faculty: J. Gomez-Herreros´s Group
S. Hormeno et al. Biophysical Journal 100, 1996-2006 (2011).
Mechanical Properties of High GoC-content DNA with A-type base-stacking
S. Hormeno et al. Biophysical Journal 100, 2006-2015 (2011).
Condensation prevails over B-A transition in the structure of DNA at low humidity"
S. E. Herrero-Galan et al. JACS 135(1), 122-131 (2013).
Mechanical identities of RNA and DNA double helices unveiled at the single-molecule level
Ares et al. Nanoscale 8,11818-11826 (2016).
High resolution atomic force microscopy of double-stranded RNA
Our group has a long-standing tradition in studying DNA-protein interactions with the AFM. In two recent collaborations we investigated how a small peptide binds and condense dsDNA using force spectroscopy methods and how Rep proteins bind to a specific region in the replication origin of plasmids.
Interactions between self-aggregating peptides and nucleic acids are underlying processes in several human diseases such us Alzheimer and Parquison. We studied an hydrophobic anticancer peptide and characterised the dynamics and mechanics of its interaction with ssDNA and dsDNA. On the other hand, we found that Rep proteins bind simultaneously to the specific dsDNA iterons region and to one of the AT-rich strands facilitating the opening of the replication bubble.
Univ. Barcelona: F. Ritort´s Group
Univ. Gdask: I. Konieczny´s Group
Univ. of Pittsburg: S. Khan and S. Leuba Groups
UCM. Biochemistry Dept.: J. Perez-Gil´s Group
CSIC. CIB: M. Oliva´s Group
J. Camunas-Soler et al. ACSnano 7 (6), 5102-5114 (2013).
Electrostatic binding and hydrophobic collapse of peptide-nucleic acid aggregates quantified using force spectroscopy"
Wegrzyn et al. Nucleic Acids Res. In press 16 MAY (2014).
Sequence-specific interactions of Rep proteins with ssDNA in the AT-rich region of the plasmid replication origin
Pastrana* Carrasco* et al. Nucleic Acids Research 44(18), 8885-8896 Published Online Aug3 (2016).
Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules
DNA origami allows fabrication of tailored nanostructures with multiple applications in nanotechnology.
In a collaborative work with groups in Dresden and Cambridge, we have used the AFM to image under liquid
conditions DNA-origami nanometric structures. These structures have been further combined with nanocapilaries
to form hybrid nanopores.
Univ. Cambridge: U. Keyser´s Group
TU Dresden: R. Seidel´s Group
N.A.W. Bell et al. Lab on a Chip 13, 1859-1862 (2013).
Multiplexed ionic current sensing with glass nanopores
S. Hernández-Ainsa et al. ACSnano 7 (7), 6024-6031 (2013).
DNA Origami Nanopores for Controlling DNA Translocation
Gollnick et al. Small 11(11), 1273-1284 (2015), (Accepted, Oct 7, 2014). (cover article).
Probing DNA helicase kinetics with temperature controlled magnetic tweezers
We are very interested in developing new technologies to increase the speed of imaging of AFMs operated in liquid environment. To do that, we initiated a collaboration with Dr. Julio Gomez Lab and Nanotec. Our approach involves modification of the AFM head to use small cantilevers; to use faster scanners; and to program faster adquisition boards. We aim to reach a realistic imaging rate of 1 frame per second on DNA in buffer.
UAM: J. Gomez´s Group
Nanotec: Adriana Gil, Pablo Ares