PROBING PROTEIN-NUCLEIC ACID INTERACTIONS WITH OPTICAL MANIPULATION
Embargo until
2023-12-01
Date
2019-08-19
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Publisher
Johns Hopkins University
Abstract
Studies of protein-nucleic acid interactions have received much attention and have revealed the sophisticated control of complicated bio-molecular systems within the cell. Among them, the careful regulation of single-stranded nucleic acids (ssNAs) is critical to maintain the integrity of genomic information. Recent advances in single-molecule techniques expanded the understanding of such dynamics with real-time observation and quantitative analysis. Here, we continue this effort by using two methods of optical manipulations to study protein-nucleic acid interactions (RNA helicase, single-stranded binding protein (SSB), and DNA helicase). External optical manipulation is applied through a hybrid platform – the combination of total internal reflection fluorescence microscopy with a single-beam optical trap. Our home-built hybrid instrument allows us to study the translocation of non‐structural protein 3 helicase (NS3h, RNA helicase) of hepatitis C virus (HCV) and the diffusion of Saccharomyces cerevisiae (S. cerevisiae) replication protein A (RPA, SSB) on long, stretched ssNAs. We observed that NS3h translocates about three times faster on ssRNA than ssDNA, and repetitive looping of DNA mediated by NS3h was reported for the first time. As for S. cerevisiae RPA, we found that the diffusion coefficient is similar or slightly higher than that of human RPA diffusion on short ssDNA, decreased with force, and was higher for the low salt conditions. To develop a bio-molecular tool for future in vivo cellular biology studies, we designed light-sensitive helicases by inserting a photo-switchable motif into Escherichia coli helicase (DNA helicase) as an internal optical manipulation based on structure-function relationship. Two light-sensitive helicases were synthesized and have shown light-induced conformational changes as a function of ATP analogues representing various intermediates of the ATP hydrolysis cycle that would occur during DNA translocation and unwinding. We also characterized the light-dependent modulation of DNA unwinding activity of these light-sensitive helicases.
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Keywords
Helicase, optogenetics, protein engineering, photoswitch, NS3h, optical trap, smFRET, Rep helicase