RUI: Characterizing DNA target search using single molecule methods

This project will investigate how proteins find specific locations in DNA. Since there are over three billion base pairs in the human genome, this is akin to finding a needle in a haystack. A variety of methods will be employed to investigate how proteins combine sliding, hoping and jumping along DNA into a search strategy.

In one method, the time taken by the protein to find its target site will be measured. In another, the motion of individual proteins during search will be tracked in real time. Stretching or coiling the DNA during the search process will demonstrate how the different conformations of DNA in the cell affect the search.

By varying the composition of the solution containing DNA and proteins, the effects of different cellular components can be determined. In another investigation, how so called "off-target" sites (sites that are similar to the target site and typically only vary by one or two base pairs) slow or perhaps even speed up the search will be studied. The laboratory work will be carried out by undergraduate students and concepts from this research will be included in courses taught by the principal investigator.

In addition, the research team will hold summer day camps for students from elementary and middle schools in Boston, where these students will be exposed to research and the scientific method.

This project will investigate search strategies of DNA binding proteins using single molecule methods. The model system chosen, restriction endonucleases, form part of the bacterial innate immune response and cleave foreign DNA at specific sites 4-8 base pairs in length. In one technique, micro beads tethered with DNA will be used to measure the exact time of cleavage of individual DNA molecules.

Using this method, hundreds of DNAs can be measured in a single experiment to yield high resolution kinetic data. A second technique will use fluorescence to track the motion of individual proteins during search. One area of investigation will characterize the roles of sliding, hopping and jumping.

The use of DNA roadblocks will distinguish between sliding, which will be blocked, and hopping, which can bypass roadblocks. Varying the conformation of DNA during single molecule imaging can test for the presence of enhanced jumping during coiling. Another area concerns off-target binding sites, which can slow target search, but also serve as reservoirs that can play a role in genetic regulation by transcription factors.

By inclusion of star sites (non-cognate sites differing by a single base pair) in DNA, quantitative models of how the position and number of such sites affect search rates can be tested. A final area involves the role of macromolecular crowding. By introducing crowding agents, the effects both on target search rates (in bead tethering experiments) and on linear diffusion along DNA (in single molecule fluorescence tracking) can be addressed.

Broader impacts of this project include training of undergraduate students, developing modules on tethered DNA for undergraduate courses, as well as a summer day camp for elementary and middle school students.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.