Mechanistic investigation of processive and distributive DNA modification

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With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Norbert Reich from the University of California at Santa Barbara to understand how proteins scan DNA in order to find specific sites for action. This process is essential to all known cellular systems. A new idea about how this happens is that enhanced motion over large distances requires DNA looping. This highly efficient approach is investigated and modeled using a newly developed theoretical approach. The implications of this work are quite broad as this means of movement is likely to be used by various proteins. This project also supports the development of a university course focused on teaching STEM students how to design experiments through extensive lab work as well as lectures and discussions. Furthermore, the expansion of a large outreach program is proposed (SciTrek) which reaches thousands of K-12 students by bringing trained university students into the classroom for several weeks to guide self-directed investigations on diverse topics. In studying the bacterial DNA adenine methyltransferase (Dam, modifies 5'-GATC-3'), it was noted that the ability to rapidly modify two or more sites is enhanced when the sites are separated up to 500 bp. Furthermore, current models of processive and distributive DNA modification fail to fully account for the efficient search processes by Dam and other proteins. A new understanding of the underlying mechanism is needed. Aim 1 attempts to answer the question: Do proteins relying on different mechanisms display distinct trajectories away from the DNA? Sliding, hopping, intersegmental transfer and intersegmental hopping mechanisms predict distinct protein movements away from the DNA and anovel experimental approach to address this is proposed. The second aim attempts to answer the question: What is the mechanism of intrasite processivity? How widespread is this activity? Dam as well as several other DNA modifying enzymes modifies both strands of DNA within the same recognition site, without dissociating (intrasite processivity). The third aim attempts to answer the question: Is efficient multisite DNA modification important in vivo? The fourth aim develops and applies quantitative theoretical/numerical tools for analyzing activity-based kinetic assays of DNA modifying enzymes. Particular emphasis is placed on elucidating the behavior of Dam, but additional systems (CcrM, uracil DNA glycosylase) are also studied to test and validate the approach more generally.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.