Evolved DNA contacts required for hexameric helicase unwinding

Baylor University, Waco TX

Investigators

Abstract

Hexameric DNA replication helicases are structurally conserved and essential enzymes present throughout all domains of life including most viruses. They utilize a common topological strategy of encircling one strand of duplex DNA to physically separate the duplex by coupled ATP hydrolysis, which results in separated single-strand DNA templates for leading and lagging strand DNA synthesis. Earlier research in the field has focused on DNA contacts within the central channel of these helicases, while recently identified exterior contacts with the excluded DNA strand have largely remained unexplored. This project will assess the importance of external helicase contacts in controlling the speed of DNA replication through direct or indirect interactions with the excluded strand. Because a multitude of hexameric helicases across all domains of life will be compared for the nature of the interactions and the consequences of disrupting them, both in vitro and in vivo, the scientific scope and impact is expected to be broad and influential. Undergraduate and graduate students will be trained in techniques of single molecule fluorescence, advanced enzyme kinetics and precise genetic manipulation. Scientific outreach programs will encourage local elementary school students to explore the wonders of their own genome through "DNA Days" and engage students in electromagnetism by building their own working audio speakers. The mechanism of DNA unwinding by hexameric helicases during genome replication currently focuses solely on interactions with the encircled strand and overlooks the implication of contacts with the excluded strand. Preliminary work suggests that these external contacts could be more influential in controlling the speed of replication, coupling enzymes at the replication fork and sensing DNA damage. This project will interrogate and quantify precise chemical specificities of excluded strand interactions, and monitor mutational consequences on replisome speed, enzymatic coordination, and genomic stability, both in vitro and in vivo. The resulting data, methods, and findings will be broadly distributed within the scientific community and will provide further insights and comparisons into the precise mechanisms of action of hexameric helicases in DNA replication across all domains of life. This project is funded jointly by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate for Mathematical and Physical Sciences.