In addition to method development, this work will shed light on important problems ranging from the fundamental biophysics of DNA-small molecule interactions to the biophysical and biochemical mechanisms of complex protein interactions involved in DNA replication and recombination. The following three specific aims will be addressed to accomplish the above goals.
To probe the binding mechanism of single-stranded DNA binding proteins, which are essential components of DNA replication. To investigate the role of important DNA replication and recombination proteins from the bacteriophage T4 system.
The PI has developed innovative biophysical methods for examining DNA binding in exquisite detail using single molecule manipulation. The results will have a significant impact beyond this field; as such methods may be adapted to examine a wide variety of important biological systems. The PI also has a strong track record of training graduate and undergraduate students in interdisciplinary research at the interface of physics, chemistry, and biology, including active collaborations with minority-serving and undergraduate institutions.
The broader impacts of this research also extend into the classroom, where the PI has developed a new Advanced Physics Laboratory course, which contains several advanced research level experiments, including the use of optical tweezers and atomic force microscopy. Experiments developed by the PI are made publicly available for use by scientists at other institutions. The PI also makes significant contributions to undergraduate teaching, bringing examples from his research into the classroom in courses such as Physics for Pharmacy and Physics for the Life Sciences.
Some full text articles may not yet be available without a charge during the embargo administrative interval. Some links on this page may take you to non-federal websites. Their policies may differ from this site. McCauley, M. Pant, K.
Nano-Bio-Photonics Laboratory | Yuval Garini's laboratory, Bar-Ilan University.
Paramanathan, T. Shokri, L.
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Stewart-Maynard, K. Williams, M. Micah J. McCauley and Mark C. Jingyun Zhang, Micah J. McCauley, L. Williams, and N. Mark C.
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Williams, Ioulia Rouzina, and Micah J. Dominic F. Qualley, Kristen M. Gorelick, Ioulia Rouzina, Mark C. Williams and Karin Musier-Forsyth. Kathy R. Chaurasiya, Thayaparan Paramanathan, Micah J. Typical FD curves corresponding to different types of complexes. Finding an optimum concentration of tethered DNA molecules on the surface is important to maximize the frequency that a single DNA molecule is picked up in a single tip approach and retract measurement.
Thus, pulling events at 3 different DNA concentrations 5.
Research Associate in Single-Molecule Biophysics
Note that the pulling efficiency, i. Based on this data we conclude that the surfaces prepared with 1. Analysis of pulling a single DNA molecule.
Although at a much lower resolution, in our assay we also observe a clear increase in the persistence length of the DNA upon binding of H-NS compare Fig. The break events we observe appear similar to ruptured intramolecular bridges as seen in optical tweezers measurements For comparison, a bare DNA molecule is shown in grey. Asterisks indicate breaking events we observe when we stretch such complex. We did not observe a preferred length of released DNA from such rupture events.
C The distances between rupture events as observed in Fig. The probability of finding a specific distance between ruptures P occurrence , as a function of the rupture distance. The double distribution is likely a reflection of rupturing the DNA-H-NS-DNA interactions in either a sheering or an unzipping geometry 14 with the later geometry requiring a significant lower force to disrupt. Comparison of pulling events in each measurement.
H-NS has been proposed to be involved in structuring the nucleoid and in gene regulation. Two different modes of binding have been reported: a stiffening mode and a bridging mode 13 , 15 , 22 , This result is in accordance with the observation that H-NS binding to DNA occurs either in a bridging mode or a stiffening mode, and that these two modes are switchable upon changes in environmental conditions 16 , But note that recent studies suggest that the DNA bridging mode is likely physiologically more relevant 24 , Yet, it is important to consider that the resilience of H-NS DNA interactions to force, depends strongly on the speed at which the force is applied, this was clearly shown by Dame et al.
In conclusion, we have described an AFM-based technique to measure interaction forces of DNA-protein complexes in high throughput.
This method is optimized to probe individual DNA-protein complexes in a population and to sample many complexes by fast scanning of the AFM tip over the surface. This technique is very suitable for high throughput analysis albeit at a lower resolution than other assays such as optical tweezers which typically have a lower throughput.
The method for sample preparation is relatively simple and quick compared to optical tweezers.
- Ripping Time.
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The described approach promises a wide applicability, not only to study DNA-H-NS interactions, but also for studies of binding strengths and dynamics of other DNA-binding and bridging proteins. A PCR fragment containing the hns gene mutated to encode serine instead of cysteine at position 21 was generated by PCR based site-directed mutagenesis on an E. By incorporating a stop codon directly upstream of the XhoI restriction site the encoded protein does not contain a C-terminal His-tag.
The supernatant was loaded onto a P11 column pre-equilibrated with the same buffer. The dialysate was loaded onto a pre-equilibrated heparin column GE Healthcare. The pooled peak fractions were again dialyzed against buffer B. Next, samples were rinsed with Milli-Q water and dried in a weak flow of nitrogen gas. Scans were collected at a scan line frequency of 0. Samples were then rinsed three times with TNM buffer. Experiments without H-NS were performed as a control.
All measurements were performed in TNM buffer. Using these images, the position of each point for collecting pulling data was determined. The spring constant of each lever was precisely determined for each experiment by using the thermal tuning method The resulting force was measured through the deflection of the cantilever using optical lever detection. Custom made programs, written in Labview TM , were used for analysis of force-distance F-D curves of pulling events.
These curves recorded the force applied on the AFM tip as it approaches and retracts from the surface. In short, the curves were translated along the x-axis to facilitate the superposition of F-D curves and to remove unspecific adhesion events at the beginning of the curve. Next the baseline and linear regions of the curve were removed and the contribution from the bending of the cantilever was subtracted.
The force on the cantilever increased gradually and dropped repeatedly upon rupture of protein bridges between or in the DNA fragments. A custom-written program in Matlab TM was used for analysing the force required for rupture of protein bridges. All force jumps below 25 pN were considered noise, and were not taken into account when analysing rupture forces generated by H-NS bridging.
Baclayon, M. Sampling protein form and function with the atomic force microscope. Mol Cell Proteomics 9 , — Heller, I. Optical tweezers analysis of DNA-protein complexes. Chem Rev , — Neuman, K. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy.
Nat Methods 5 , — Sitters, G. Acoustic force spectroscopy.