| NSF Org: |
MCB Division of Molecular and Cellular Biosciences |
| Recipient: |
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| Initial Amendment Date: | June 26, 2017 |
| Latest Amendment Date: | June 26, 2017 |
| Award Number: | 1715317 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Wilson Francisco
wfrancis@nsf.gov (703)292-7856 MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences |
| Start Date: | August 1, 2017 |
| End Date: | July 31, 2021 (Estimated) |
| Total Intended Award Amount: | $340,471.00 |
| Total Awarded Amount to Date: | $340,471.00 |
| Funds Obligated to Date: |
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| History of Investigator: |
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| Recipient Sponsored Research Office: |
400 FENWAY BOSTON MA US 02115-5725 (617)735-9979 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
400 The Fenway Boston MA US 02115-5725 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Molecular Biophysics |
| Primary Program Source: |
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| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.074 |
ABSTRACT
This project will investigate how protein molecules are able to find specific locations in the genome accurately and quickly. Since there are over three billion base pairs in the human genome, this is akin to finding a needle in a haystack. Scientists have discovered that many proteins slide along the DNA during their search. Using highly sensitive techniques that can observe single molecules of DNA, this project will investigate how and why proteins slide. The proteins studied in this project, known as restriction endonucleases, cleave DNA into two strands once they bind to their target site. Many proteins that regulate and maintain the genome cleave DNA in their normal functioning. New techniques of genetic engineering also require cleavage of DNA at a specific site. This project will investigate how this process occurs. All laboratory work will be carried out by undergraduate students who will be trained in cutting edge technical skills as well as in scientific thinking and problem solving. Concepts from this research will be included in courses taught by the investigator. In addition, the investigator will hold workshops for students and teachers from Boston public schools. The workshops will expose high school students to scientific research and help teachers to create materials they can take back with them to use in their classrooms.
Site specific DNA cleavage is a crucial step in antibody production, the prokaryotic adaptive immune system and genome editing. This project will investigate the target search strategies and cleavage mechanisms of a model system (restriction endonucleases) using a combination of single-molecule techniques and computational modeling. In one technique, microbeads tethered with DNA will be used to measure the exact time of cleavage of individual DNA molecules. In a single experiment, hundreds of DNA can be measured to yield high resolution kinetic data. A second technique will use fluorescence to track the diffusion of individual proteins along DNA. One dimensional diffusion constants, as well as off-rates, can be determined from single particle trajectories. Theoretical models of target search will be developed and compared to experimental data. Computer simulations of random walks, as well as models based on chemical kinetics, will be explored. The PI will train undergraduate students in research, scientific communication and scientific writing. Modules on tethered Brownian motion and DNA cleavage will be introduced into a biochemistry course. In outreach activities, half day research experiences for Boston area high school students will be held. In addition, a professional development course for high school teachers will be created.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
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PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
In this project, a research group made up of undergraduate students and the principle investigator collected data, developed models and created new methods for studying the process of target search by DNA binding proteins. Target search is the critical first step in any interaction between the cell and its genome. Our data has strengthened the developing view on how proteins combine 3D and 1D searches, quantified how 1D searches are carried out, and discovered new twists in the process. Specifically, we have validated the consensus model of 1D diffusion (including sliding/hopping in combination) in a new way. Our data shows a transition between sliding and a mode that combines sliding and hopping. We developed a new method for determining the sliding/hopping ratio quantitatively. Our experiments lead to the novel observation of pausing during target search. We were able to map pause sites in the target genome and identify the genetic signature of these sites, which differ in a single base pair from the target site. We also measured the DNA length and salt dependence of target search. With our data, we evaluated competing models and were able to propose a simple model combining 1D and 3D search that agreed with our data. In addition, we developed a novel method to block sliding on DNA using dCas9 as a programmable roadblock. Our data showed that nearby roadblocks slow target search by NdeI is a salt dependent manner, further supporting our model of target search.
The experimental and theoretical models created, including QD labeled EcoRI, flow stretching TIRF single molecule tracking, bead loss based target search time measurements, and biochemical models of target search will allow future studies of both 1D target search, the role of off-target interactions, as well as studying the effects of more cellular like conditions.
In this project, ten undergraduate students were trained in cutting edge biophysical laboratory techniques. They authored four scientific papers and presented their work at four national scientific meetings. Eight of these students have graduated and are either working in biotechnology or pursuing post graduate training in science or medicine. Additionally, the results of this research have been translated into educational materials and disseminated to benefit undergraduate science education in biophysics.
Last Modified: 08/05/2021
Modified by: Allen Price
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