Helicases are molecular motors that couple nucleotide binding and hydrolysis to nucleic acid unwinding and translocation. Many organisms encode multiple helicases that are essential to fundamental cellular functions such as DNA replication, repair, recombination, transcription and translation. Several human genetic disorders have also been linked to mutations in DNA helicases and helicases from human pathogen are being actively pursued as drug target. We propose to study the molecular mechanisms of two classes of helicases using the powerful single molecule fluorescence techniques. Our approaches have already yielded several surprises and provided previously unattainable data on complex biochemical processes. For example, using single molecule fluorescence resonance energy transfer (FRET), we discovered that PcrA helicase reels in DNA in single base steps, forming a DNA loop, and at the same time efficiently removing other proteins bound to the DNA (Park et al, Cell, 2010). We also discovered that NS3 helicase from a human pathogen, hepatitis C virus, unwinds DNA using a spring-loaded mechanism (Myong et al, Science, 2007). On superfamily 1 helicases (Rep/UvrD/PcrA), we aim to (1) uncover the mechanism and functional importance of chemo-mechanical coupling between ATP hydrolysis and DNA translocation, (2) correlate DNA unwinding and enzyme conformational changes using a hybrid instrument combining ultrahigh resolution optical tweezers with single fluorophore detection, (3) probe consequences of an encounter between a helicase and other DNA bound proteins via imaging-based localization. On replicative hexameric helicases, we aim to (1) determine the step size of DNA unwinding and inter-subunit coordination of T7 gp4, DnaB-like helicase G40P and SV40 Large T antigen (LTag), (2) investigate the mechanism of helicase slipping and how other interacting proteins modulate the slipping events, (3) test if the ring-shaped helicase rotates around the DNA axis during its translocation along double stranded DNA, and (4) probe for a potential coordination two hexamers of LTag unwinding from the origin sequence in opposite directions using four-color single molecule FRET. In all of our studies, bulk-phase biochemical experiments and structural data through collaboration will help us design and interpret our measurements.
Several human genetic disorders have been linked to mutations in DNA helicases and helicases from human pathogen are being actively pursued as drug target. This application is focused on two broad classes of DNA helicases, superfamily 1 (SF1) helicases and hexameric helicases, that function in DNA repair and replication, respectively. Since DNA replication and repair are fundamental to cell growth in all organisms, an understanding of such a basic process as enzyme-catalyzed DNA unwinding will undoubtedly have an impact on our understanding of some cancers that result from defects in replication or repair.
|Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei et al. (2016) Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation. Nat Commun 7:11045|
|Ngo, Thuy T M; Yoo, Jejoong; Dai, Qing et al. (2016) Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability. Nat Commun 7:10813|
|Ha, Taekjip (2016) Probing Nature's Nanomachines One Molecule at a Time. Biophys J 110:1004-7|
|Chowdhury, Farhan; Li, Isaac T S; Ngo, Thuy T M et al. (2016) Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo. Nano Lett 16:3892-7|
|Brenner, Michael D; Zhou, Ruobo; Conway, Daniel E et al. (2016) Spider Silk Peptide Is a Compact, Linear Nanospring Ideal for Intracellular Tension Sensing. Nano Lett 16:2096-102|
|Singh, Digvijay; Sternberg, Samuel H; Fei, Jingyi et al. (2016) Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat Commun 7:12778|
|Arslan, Sinan; Khafizov, Rustem; Thomas, Christopher D et al. (2015) Protein structure. Engineering of a superhelicase through conformational control. Science 348:344-7|
|Kim, Sung Hyun; Park, Jeehae; Joo, Chirlmin et al. (2015) Dynamic growth and shrinkage govern the pH dependence of RecA filament stability. PLoS One 10:e0115611|
|Zhang, Jichuan; Fei, Jingyi; Leslie, Benjamin J et al. (2015) Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells. Sci Rep 5:17295|
|Comstock, Matthew J; Whitley, Kevin D; Jia, Haifeng et al. (2015) Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme. Science 348:352-4|
Showing the most recent 10 out of 121 publications