DNA Strand Separation: A New Mechanism of DNA Recognition

University Of California-Santa Barbara, Santa Barbara CA

Investigators

Abstract

With the support of the Chemistry of Life Processes (CLP) program in the Division of Chemistry, Professor Norbert Reich of the University of California, Santa Barbara is studying a new DNA recognition mechanism reliant on the separation of DNA strands. This project has the potential for broad impact since DNA recognition is fundamental to all of life. Furthermore, the training of graduate and undergraduate students in diverse biochemical, chemical, and biological methods, will enable their entry into the biotechnology industry. The proposal also supports the science outreach, SciTrek, which brings university students into local classrooms wherein K-12 students are guided in multi-day inquiry of diverse (science, technology, engineering and mathematics) STEM topics. This program has been shown to impact students from underrepresented groups and increase their interest in STEM; a similar impact on participating girls has been documented. An additional 600 K-12 students are expected to be participate in such outreach sessions over the period of the award. The bacterial DNA adenine methyltransferase, CcrM, is extremely discriminating in the sequences that undergo methylation, with the double-stranded site 5’-GANTC-3’ being its normal target. The enzyme is broadly dispersed in diverse bacteria, many of which are human pathogens. The mechanism whereby CcrM induces the strands to become separated is not known, and although used by enzymes such as CRISPR, the entire process remains obscure. Understanding DNA recognition is fundamental to life, and has implications for the development of highly selective inhibitors that disrupt this process. The project will rely on protein engineering of amino acid residues implicated in the protein-DNA co-crystal structure to be important for the strand-separation process. Fluorescence-based assays of conformational changes have been developed and will be applied to these mutants. It is anticipated that the proposed work will help to elucidate how CcrM induces strand separation and will provide tools for the study of this mechanism in other enzymes. 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.