In this CAREER proposal the PI proposes to study the mechanisms of DnaB replication using single molecule manipulation measurements of the DNA activity. The specific scientific aims of the proposal are: 1. Determine the mechanism of DnaB translocation. 2. Determine the mechanism of dsDNA unwinding. (The PI will study this question by altering the fraying equilibrium of dsDNA through application of a destabilizing mechanical force, and measuring the resulting unwinding velocity.) 3. Develop a novel single-molecule technique that will allow multiple single-molecule measurements to be performed in parallel. The results of the proposed research will lay the groundwork for future studies of the mechanistic behavior of the entire replication complex and will be relevant for understanding replication dynamics in higher organisms. The educational aim of the proposal is to train the next generation of interdisciplinary, life science oriented researchers by introducing undergraduate and graduate students from the physical sciences to biological probes. This will be accomplished through both laboratory internships integrated with the research aims of this proposal, and through classroom teaching. The PI will train both graduate and undergraduate students, who will learn techniques from biology, biochemistry and physics in order to carry out the experiments. Participating undergraduate students will be recruited from several backgrounds, including those that are underrepresented in the sciences.
In all organisms, the DNA molecule(s) that comprise the genome must be faithfully copied. This is done by a complex of molecular machines termed the replisome. A key member of this complex is the helicase, a machine that sits at the head of the complex, and is responsible for opening the DNA double helix for copying. In this project, we developed and applied tools that permitted the study of the microscopic mechanisms of helicases from multiple organisms. Our major outcomes, in terms of intellectual merit, were the following: 1) We developed novel methodology permitting the high-speed, high-resolution, yet multiplexed, study of single biomolecules. This technique is broadly applicable to many biomolecular systems; indeed, it has been widely adopted by the community since we developed it. 2) We found that the bacterial replicative helicase, DnaB, moves along DNA through a 'scrunching' mechanism involving local DNA compaction, and that its speed is determined by the geometry and tension on the DNA strands it interacts with. We found that a putatively similar helicase, T4 gp41, also had a geometry/tension dependence, but one that was opposite to that of DnaB. This indicates an unexpected diversity in mechanism between helicases thought to be quite similar. This result is of utmost importance in our understanding of replication, and could point to novel ways that the replication complex interacts with, and responds to, damaged DNA. 3) Our work involved application of force to single DNA molecules; in doing so, we found an unexpected elastic response of those DNA molecules. This measurement validated a previously-unproven theory of the physics of polymers. Our major outcome, in terms of broader impact, has been the training of multiple young researchers in a highly-interdisciplinary field: Four graduate students, and over 10 undergraduates, have been supported wholly or partially by this award, and they have learned a great deal of physics, chemistry, biology, and instrumentation. This corps of trained scientists have continued in various scientific/engineering endeavors, and are having a wide impact on society through their efforts.
