Progress 10/01/00 to 09/30/05
Outputs
We used high resolution Atomic Force Microscopy (AFM) to image the compaction of linear and circular DNA by the yeast mitochondrial protein Abf2p, which plays a major role in packaging mitochondrial DNA. AFM images show that protein binding induces drastic bends in the DNA backbone for both linear and circular DNA. At a high concentration of Abf2p DNA collapses into a tight globular structure. We quantified the compaction of linear DNA by measuring the end-to-end distance of the DNA molecule at increasing concentrations of Abf2p. We also derived a polymer statistical mechanics model that provides a quantitative description of compaction observed in our experiments. This model shows that sharp bends in the DNA backbone are often suffcient to cause DNA compaction. Comparison of our model with the experimental data showed excellent quantitative correlation and allowed us to determine binding characteristics for Abf2p. These studies indicate that Abf2p compacts DNA through
a simple mechanism that involves bending of the DNA backbone. We discuss the implications of such a mechanism for mitochondrial DNA maintenance and organization. Mitochondria participate in many critical processes in the cell lifecycle. Aside from their primary role in ATP production, mitochondria also act as signaling centers through the regulation of calcium, iron and metabolite levels in the cytosol. These organelles are also responsible for the main switch controlling apoptosis. Such critical responsibilities place stringent requirements on the integrity of the mitochondrial DNA (mtDNA). A variety of processes threatens mtDNA. The respiratory chain of mitochondrial metabolism produces large levels of oxygen radicals which can attack mtDNA. Oxidative damage to mtDNA often leads to several clinical disorders including Parkinson's, Hutchinson's, and Huntington's disease. Ironically, the very job that is required to keep the cell alive also yields dangerous byproducts. In order to
operate under these harsh conditions mtDNA must be packaged in a way that protects it from damage, while not impairing the normal functions of mtDNA such as replication and transcription. Mammals (Van Tuyle and McPherson, 1979; Satoh and Kuroiwa, 1991) and the budding yeast S. cerevisiae (Wintersberger et al., 1975; Miyakawa et al., 1984, 1987) package mtDNA in compactglobular structures similar to a bacterial nucleoid. These mt-nucleoid structures are distinctly different from the packaging of DNA into chromatin in the cell nucleus. Researchers have firmly established the mechanism of histone protein action in the packaging of nuclear DNA (Luger et al., 1997). However, very little data exists on the identity or function of the proteins that facilitate the formation of the mt-nucleoid. Diffley and Stillman found that a particular 20 kDa protein was present in relatively high abundanceamong the various polypeptides isolated from mt-nucleoids (Diffley and Stillman, 1988). This protein,
Abf2p (ARS binding factor 2), displays interesting DNA binding characteristics: it binds non-specifically to general regions of DNA, but exhibits phased binding to replicating sequences such as ARS1.
Impacts
These studies will enable us to better understand how genetic information is handled in mitochondria. This will allow us to better understand the way mitochondria function in a cell. AFM imaging can provide quantitative characterization of protein-DNA interactions. Single molecule imaging not only can reveal the geometrical conformation of the protein-DNA complexes, but also can determine thermodynamic parameters for protein binding. We believe that AFM imaging will be an important part of the modern biophysics tool kit for studies of protein-DNA interactions. These interactions are basic to all living organisms. Second, we believe that our results will be important for establishing the mechanisms of mitochondrial DNA maintenance and regulation. The apparent loose packing of DNA by the Abf2p should provide important clues for the structure of the mitochondrial nucleoid and for possible access pathways for regulatory proteins. Further AFM studies using other
mitochondrial proteins should provide a wealth of information about maintenance and regulation of mitochondrial DNA and how this impacts human and animal health.
Publications
- Friddle, R.W., Klare, J., Martin, S., Corzett, M., Balhorn, R., Baldwin, E., Baskin, R., and Noy, A. (2004) Mechanism of DNA compaction by yeast mitochondrial protein Abf2. Biophysical Journal 86:1-8.
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Progress 01/01/03 to 12/31/03
Outputs
Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although DNA-protein complexes called 'nucleoids' have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and AFM single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (koff = .014 plus or minus .001 sec-1) and the extremely small forces (less than 0.6 pN) stabilizing the condensed protein-DNA complex. AFM images of individual complexes showed the 190 nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.
Impacts
These studies will enable us to better understand how genetic information is handled in mitochondria. This will enable us to better understand the way mitochondria function in a cell.
Publications
- Brewer, L.R., R. Friddle, A. Noy, E. Baldwin, S.S. Martin, M. Corzett, R. Balhorn and R.J. Baskin. 2003. Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p. Biophysical Journal 85:2519-2524.
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Progress 01/01/02 to 12/31/02
Outputs
ABF2p is a 20 Kda nuclear encoded protein found in the mitochondria (mt) of the budding yeast Saccharomyces cerevisiae and thought to package its genome into spheroidal 'nucleoids'. ABF2p has been shown to play important roles in mtDNA maintenance and copy number, transcription, and recombination. The ABF2p DNA binding footprint is thought to be between 25 and 30 bp and its charge is approximately +12. ABF2p is closely related in sequence and function to the vertebrate nuclear high-mobility group (HMG) protein HMG1 (9) and the mammalian mitochondrial transcription factor mtTF1. HMG proteins bind in the DNA minor groove, strongly bend DNA, and recognize four-way (Holliday) junctions. ABF2p is the most abundant mitochondrial protein, with enough protein present to cover the entire mitochondrial genome (8). We have observed the packaging of individual molecules of linear dsDNA into compact protein-DNA complexes by the yeast mitochondrial protein ABF2p using both
fluorescence microscopy and atomic force microscopy (AFM). An optical trap was used to manipulate single DNA molecules into and out of buffer containing ABF2p protein using a two-channel flow cell. We measured the on and off-rate of ABF2p protein from single DNA molecules, and verified that the binding obeyed first order kinetics. We determined the equilibrium binding constant Kb, the fractional coverage of DNA by ABF2p and the Gibbs free energy of binding, dG. We also observed that each DNA molecule was compacted by ABF2p between 50 and 100% of its initial extension and that the compaction varied inversely with the molecule's hydrodynamic drag. We show that ABF2p protein binds uniformly to an individual DNA molecule, and hypothesize that complete compaction in flow does not occur because of the stretching of the protein-DNA complex induced by the buffer. It is not clear how much ABF2p modifies the native persistence length of DNA, but 90-100% compaction was only achieved for DNA
molecules where the hydrodynamic drag of the initial bare DNA was approximately 0.6 pN or less. AFM images of single DNA molecules complexed with ABF2p revealed diffusely packaged spheroidally shaped structures. The packaging of DNA by ABF2p thus appears to be very weak.
Impacts
These studies will enable us to better understand how genetic information is handled in mitochondria. This will enable us to better understand the way mitochondria function in a cell.
Publications
- L. Brewer, A. Noy, J. Klare, J.Lengyel, M. Corzett, S. Martin, R. Balhorn, E. Baldwin, and R.J. Baskin. DNA Compaction Induced by the Mitochondrial Packaging Protein ABF2. Biophysical Society Abstracts, 2002.
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Progress 01/01/01 to 12/31/01
Outputs
NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although, NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD over 2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NODDTW and NODDR2) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport: 1) NOD does not produce microtubule gliding; 2) the Kd of NOD for microtubules is weaker in the presence of ATP than in the presence of ADP; and 3) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of
chromosomes to microtubules rather than producing vectorial transport.Kinesins convert the chemical energy stored in ATP into mechanical energy for unidirectional transport along microtubules (MTs) (Brady, 1985; Block et al., 1990; Romberg & Vale, 1993; Crevel et al., 1996; Lockhart & Cross, 1996; Rice et al., 1999; Vale & Milligan, 2000). This generation of force is based on a cycle of conformational changes, dependent upon the hydrolysis state of the bound nucleotide. The hydrolysis state of the nucleotide appears to be monitored by two regions which interact with the gamma phosphate of ATP called switch I and switch II (Vale and Milligan, 2000). These gamma phosphate sensors are found in G-proteins, myosins and kinesins (Vale and Milligan, 2000). The phosphate sensor moves in response to phosphate release, and this is transmitted and amplified by the switch II helix to other parts of the motor protein. Switch II is involved in communication between the active site, the allosteric
activator (polymer binding site for kinesin and myosin), and the mechanical elements (kinesins and myosins). It is not clear which microtubule binding regions underlay changes in microtubule affinity in various nucleotide states.
Impacts
These studies relate to the way cell division is controled in all plants and animals. An understanding of how the protein NOD works will allow us to better understand how meiosis occurs.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
RecBCD enzyme is a processive helicase and nuclease that participates in the repair of chromosomal DNA via homologous recombination. Visualization of translocation and DNA unwinding by single DNA helicase molecules permits study of the stochastic properties of individual molecular motors, or "nano-machines", that is obscured in the population-average of steady-state, bulk phase measurements. We have directly visualized the movement of individual RecBCD enzymes on single molecules of duplex DNA (dsDNA). DNA substrates were constructed by attaching a biotinylated oligonucleotide to one cohesive end of lambda DNA. Detection involves optical trapping of solitary, fluorescently-tagged dsDNA molecules that are attached to polystyrene beads and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by displacement of fluorescent dye from the DNA by the enzyme. Under these conditions,RecBCD enzyme will bind to the end of
dsDNA but it will neither translocate nor unwind DNA. These helicase-DNA complexes were introduced into one channel of a Y-shaped, micro-machined flow cell. Helicase reaction buffer containing ATP was introduced into the second channel under conditions of laminar flow, creating a situation where the two solutions flowed parallel to one another with minimal mixing. Unwinding is both continuous and processive, occuring at a maximum rate of 972+/- 172 bp/sec (0.30 um/sec) with as much as 42,300 bp of dsDNA unwound by a single RecBCD enzyme molecule. DNA shortening is linear and constant with no discernable pauses. For individual RecBCD enzyme molecules, both the rate and processivity can vary by several fold, but the mean behaviour corresponds to that observed in bulk solution.
Impacts
These are the first studies of a single (helicase) enzyme molecule interacting with a single molecule of DNA. No one has previously visualized this process
Publications
- Bianco,Brewer,Corzett,Balhorn,Yeh,Kowalczykowski, and Baskin. Processive Translocation and DNA Unwinding by Individual RecBCD Enzyme Molecules; Nature, In Press (2000).
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Progress 01/01/99 to 12/31/99
Outputs
Our molecular model has been applied to the Kinesin-Tubulin system. Based on the dimeric structure of kinesin we assume that one head of kinesin is strongly bound to a beta-tubulin subunit. Behind this "front" head is a second head which, due to structural considerations, is oriented differently and is only able to weakly bind to a beta-tubulin subunit. The front head is oriented toward the plus end of the micro tubule and both heads move along a single proto-filament. At the completion of a single ATP hydrolysis cycle, the front head releases from binding to the beta-tubulin substrate, the protein tether extends and the head diffuses along the protofilament. The second heads tether, not being strongly bound, allows the first head as well as the cargo to diffuse along the protofilament. (The weak binding structure might be a kinesin-ADP complex.) The first head diffuses until it finds the next binding site, 8nm from the first, and is able to strongly bind thus
effectively shortening the protein chain tether by 5-6 nm and pulling both the cargo and the weakly bound second head along the protofilament. In this model the first (front) head is only capable of strong binding and the second head is only capable of weak binding. The step size is 8 nm and the "stroke" size is 5-6 nm. Weak binding (protein friction) holds the motor onto the tubulin substrate. ATP provides the energy to create a structure in the enzyme that is able to change by induced fit into the substrate. (In this model all binding interactions are with the beta-subunit of tubulin but minor interactions with alpha-tubulin are not precluded by this model.)
Impacts
We presented a model in which the front head of the motor is the working one, and the rear head is idle, playing a directional role. We discuss structural data and demonstrate that the model explains mechanical, kinetic and statistical experimental data.
Publications
- Collins, S., Baskin, R.J. and Howitt, D.G. 1999. Micro instrument gradient-force optical drap. J. Applied Optics 38:6068-6074.
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Progress 01/01/98 to 12/01/98
Outputs
We have developed a model of a molecular motor in which three factors combine to provide a basis for sequential movement. In this model, a motor head, tethered by a length of protein chain, diffuses to a site on the substrate capable of strongly binding to the catalytic core of the motor. As binding occurs, the fit of the enzyme to the substrate induces a change in the motor structure such that the protein tether is drawn into the body of the catalytic core thus effectively shortening the tether length by 5-6 nm. This induced fit shortening of the catalytic core tether is, in effect, a power stroke and pulls the motor cargo in the direction of motion of the enzyme. In this model we assume that a second motor head is weakly bound to another substrate binding site serves to hold the motor in proximity to the substrate thus reducing the diffusional freedom of the first head. This second motor head is identical in structure to the first head but, due to structural
constraints, is not able to strongly bind to the substrate. It also contains a protein tether but in the absence of strong binding, this tether is not tightly held to the catalytic core. The weak binding of this second head (protein friction) is disrupted during the "power stroke" of the first head, and the second head is pulled behind the first until it again weakly binds at a further site along the substrate. The net movement of cargo of 7-8 run will occur if strong binding sites are spaced at about 8 nm and if the cargo is moved about 2 run by Brownian diffusion.
Impacts
(N/A)
Publications
- COLLINS, S., KNOESEN, A. and BASKIN, R.J. 1997. Micromachined optical trap for use as a microcytology workstation. Proc. SPIE. 2978:69-74.
- MOGILNER, A., MANGEL, M. and BASKIN, R.J. 1997. Motion of molecular motor ratcheted by internal fluctuations and non-linear protein friction. Physics Letters. 237:297-306.
- BASKIN, R.J. 1998. Design and use of the centrifuge microscope to assay force production. Methods in Enzymology. 298:413-427.
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Progress 01/01/97 to 12/01/97
Outputs
Kinesin, myosin and RNA polymerase, moving along microtubules, actin filaments and DNA, respectively, are examples of biologically important families of molecular motors. In each example, proteins move unidirectionally along the track (i.e. protein or nucleic acid). The central question about these motor proteins is to characterize the mechanochemical transduction which generates a directed force and results in protein movement. Recently, theoretical modeling has proved to be a valuable tool in understanding the action of motor proteins. Thermal ratchet models are based on rectifying Brownian diffusion of molecular motor by either periodic potentials asymmetric in space (which can be generated by periodic array of dipoles) or by force with a zero mean value asymmetric in time. Power stroke models ascribe the motion of the motor to the conformational change in the motor induced by nucleotide binding and/or hydrolysis or to binding to the track. In all models, the
existence of an effective potential periodic in space in which the protein moves is used explicitly. We have demonstrated how a power stroke model can explain the unidirectional motion of the motor protein without postulating an effective periodic potential. The necessary rectifying mechanism in this case is (weak) irreversible visco-elastic binding of the molecular motor to the track.
Impacts
(N/A)
Publications
- HALL, K., COLE, D., YEH, Y. and BASKIN, R.J. 1996. Kinesin force generation measured using a centrifuge microscope sperm-gliding motility assay. BiophysicalJournal 71:3467-3476.
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Progress 01/01/96 to 12/30/96
Outputs
To measure force generation and characterize the relationship between force and velocity in kinesin-driven motility, we have developed a centrifuge microscope sperm-gliding motility assay. The average (extrapolated) value of maximum isometric force at low kinesin density was 0.90/+-/0.14 pN. Furthermore, in the experiments at low kinesin density, sperm pulled off before stall at forces between 0.40 and 0.75 pN. To further characterize our kinesin-demembranated sperm assay, we estimated maximum isometric force using a laser trap-based assay. At low kinesin density, 4.34/+-/1.5 pN was the maximum force. Using values of axoneme stiffness available from other studies, we concluded that, in our centrifuge microscope-based assay, a sperm axoneme functions as a lever arm, magnifying the centrifugal force and leading to pull-off before stall. In addition drag of the distal portion of the axoneme is increased by the centrifugal force (because the axoneme is rotated into closer
proximity to the glass surface) and represents an additional force that the kinesin motor must overcome.
Impacts
(N/A)
Publications
- HALL, K., COLE, D., YEH, Y. and BASKIN, R.J. 1996. Kinesin force generation measured using a centrifuge microscope sperm-gliding motility assay. BiophysicalJournal 71:3467-3476.
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