Coordination of complex dynamics by DNA manipulating enzymes

University Of Missouri-Columbia, Columbia MO

Investigators

Abstract

Project Summary My laboratory will use a combination of magnetic tweezers-based force and torque measurements, single molecule fluorescence measurements, and computational methods to study the activity of two genome maintenance systems: (1) the human transcription factor IIH (TFIIH) and (2) the type IA topoisomerase of mycobacteria. Both of these systems require the coordinated movements of multiple components to accomplish their functions. We will make use of two different single molecule methods to simultaneously measure orthogonal degrees of freedom. Using this approach we will be able to follow complex protein-DNA dynamics in real-time in order to better understand these systems. We will also use molecular dynamics simulations to determine the molecular motions underpinning the experimental observations. TFIIH is a DNA unwinding enzyme that plays a major role in two of the most important cellular functions, transcription of DNA to RNA and repair of damaged DNA. Because of this dual function, TFIIH is a potential target for chemotherapeutics. It has also been linked to human disorders of DNA repair. TFIIH is a large, multidomain protein complex. The core TFIIH complex includes both a DNA helicase domain and a DNA translocase domain. Evidence from biochemical studies and high resolution cryo-EM structures suggest that in the nucleotide excision repair pathway, these domains work in concert to open a bubble in DNA. A major limitation of such studies, however, is the inability to directly observe the active dynamics of the enzyme on the structure of DNA. Over the next five years, we will develop and conduct simultaneous single molecule force and fluorescence assays to observe TFIIH in action to understand how the helicase and translocase domains coordinate their activities. Type IA topoisomerases are a class of enzyme that are found in virtually all organisms. These enzymes remove excess supercoils from DNA to maintain genome integrity. While many chemotherapy drugs and antibiotics target other topoisomerases, there are currently no drugs in use against type IA topoisomerases, making them an important potential target for novel therapeutics. The type IA topoisomerase of Mycobacterium tuberculosis has been identified as a potential target for antibiotics against drug-resistant tuberculosis. Mycobacteria type IA topoisomerases contain unique C-terminal domains which are required for passage of one strand of dsDNA through a protein-mediated DNA-gate in the other strand. The details of this strand passage activity and its coupling to gate opening are unclear. We will use a combination of single molecule force and fluorescence assays and molecular dynamics simulations to probe the activity, conformational dynamics, and drug-response of mycobacteria type IA topoisomerases. Single molecule methods offer the opportunity to understand the mechanisms of enzyme activity by following dynamic molecular processes in real-time. Combining different single molecule methods will allow us to measure different aspects of the enzyme-DNA interactions, deepening our understanding of these processes.