Mechanisms of DNA helicases and their regulation

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PROJECT SUMMARY / ABSTRACT “Mechanisms of DNA helicases and their regulation” Helicases are a ubiquitous and diverse group of molecular machines that separate the strands of nucleic acids. They are essential actors in many genome maintenance processes in all domains of life, including some viruses. As a result, helicases are biomedically important proteins, and their pathologies are associated with a number of human diseases and cancer. Since uncontrolled unwinding is detrimental to genomic integrity, helicase activity must be tightly regulated in the cell. Furthermore, since many helicases are able to play multiple, distinct roles in a variety of cellular pathways, they must be activated only in the correct contexts. How these different functions are defined and regulated remains poorly understood. In this project, we will investigate the molecular mechanisms by which DNA helicases are regulated. Our studies will focus on the model non-hexameric helicases UvrD, Rep, and XPD, which are critical components of the cellular response to DNA damage in prokaryotes, eukaryotes, and archaea and also serve as prototypical members of the two largest structural superfamilies of helicases. Insights gained on their mechanisms are expected to extend to a number of structurally and functionally homologous systems. Prior work by us and others has shown that these types of helicases have auxiliary domains and/or make secondary contacts with DNA that play regulatory—often, auto-inhibitory—roles. Protein partners to helicases have thus been proposed to activate helicase activity by controlling these mechanisms, thus defining helicase roles in the cell. To gain insights into these mechanisms, our studies will focus on two main research goals: understanding how interactions with DNA and non-canonical DNA structures control helicase activity (Goal 1), and quantifying how encounters with accessory proteins—both protein partners that recruit and activate helicases and proteins that compete for the same DNA substrates—regulate helicases (Goal 2). Our approach for achieving these research goals will integrate advanced single-molecule biophysical techniques—optical tweezers combined with fluorescence microscopy—together with traditional biochemistry and computational biophysics methods. These approaches leverage our group's expertise and that of the assembled collaborators, and have been successfully applied by us in our high-resolution measurements of helicase unwinding and conformational dynamics, their modulation by interactions with accessory proteins, and their connection to atomic-level structural models of helicases,. Beyond providing insights on helicase mechanism and the genome maintenance pathways in which they participate, our studies will advance new biophysical methods for investigating biomolecular dynamics.