Authors:

Alicia Qin

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About Paper:

DNA topoisomerases are essential enzymes that regulate DNA supercoiling through cutting, strand passage, and resealing, which is crucial for keeping genetic material stable while copying and reading DNA. Given their pivotal role in DNA metabolism, topoisomerases have emerged as attractive targets for cancer therapies, but current inhibitors face challenges such as toxicity and resistance, highlighting the need for drug design informed by deeper understandings of enzyme dynamics. Despite detailed structural insights, the conformational changes enabling Topoisomerase | (TOP) to perform strand passage remain unclear, with models proposing various movements between its four domains. The present study explores the role of domain movements in DNA relaxation by TOP1 using a technique called single-molecule FRET (smFRET). We began with protein growth and purification using the E. coli C41(DE3) cell line. One domain of the TOP1 enzyme was labeled with a Cy3 fluorophore (a fluorescent tag) and another with a Cy5 fluorophore. We used a 561 nm laser to excite the fluorophores in a microfluidic flow cell, with DNA present in one channel and absent in the other, enabling comparison of protein behavior between the two conditions. The MATLAB-based DeepLASI software was then used to find traces where the Cy5 signal exhibited an anti-correlation with Cy3 fluorescence, confirming direct energy transfer from one domain to another due to spatial proximity and interaction. Analysis of FRET data reveals novel conformational changes within different domains as a result of DNA binding. These findings demonstrate for the first time that FRET can be used to observe TOP1 conformational dynamics. We will continue to investigate the interactions between all domains of TOP1 and ultimately provide the foundation for designing better inhibitors to overcome current therapeutic challenges.

Source:

Northwestern University

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Co-authors:

Alicia Qin