We will study conformational changes of DNA helicases and functions using novel fluorescence assays. The assays include single molecule fluorescence resonance energy transfer (smFRET), quenching, single molecule counting, and ensemble FRET. Single molecule techniques can measure helicase activities in real time free from ensemble averaging. These techniques are also applicable to many biological systems, and indeed we have used them recently to detect conformational changes in RNA molecules and to study the folding pathways and catalysis of a ribozyme. The specific helicases will be from SF1 helicase family that includes e.coli Rep and UvrD helicases. The mechanistic information obtained should facilitate studies of other helicases as well. 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. Helicases also share many properties with other molecular motors. Hence a fundamental understanding of helicases is of both scientific and medical importance. In a previous study, we have used smFRET between dyess attached to DNA to study E. coli Rep helicase. We immobilized DNA on a polymer-coated surface, which enabled extended observation time while maintaining nearly complete biochemical activity. Unwinding of only a few base pairs, hence the distance change between two dyes attached to the DNA could be detected via smFRET. We also discovered a number of new conformations and determined the fluctuation rates among them. Further work using these techniques, proposed here, is poised to answer many fundamental questions. (1) Is oligomerization of helicase necessary for DNA unwinding and if so why? (2) How many base pairs are unwound per biochemical cycle and what factors influence the unwinding processivity? How tightly coupled is ATP hydrolysis to unwinding? (3) What are the functional roles of helicase conformational change; how nucleotide and DNA binding influence them? (4) What is the origin for directionality of DNA unwinding? To achieve these goals, we will use both ensemble and single molecule measurements of dyes attached to various sites on DNA and helicase. Specifically, FRET (or quenching) will be measured between sites on DNA, between helicase and DNA, between helicase and ATP, between two sites on helicase monomer, and between two monomers. The number of helicases bound to each DNA will be determined by counting the dyes (one dye per monomer). Correlation will be made between single molecule signals detected simultaneously. For example, we will measure both the number of helicase monomers bound to DNA and DNA unwinding to determine if the active form of SF1 helicase is monomer or dimer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Lewis, Catherine D
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Illinois Urbana-Champaign
Schools of Engineering
United States
Zip Code
Singh, Digvijay; Wang, Yanbo; Mallon, John et al. (2018) Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis. Nat Struct Mol Biol 25:347-354
Figiel, Ma?gorzata; Krepl, Miroslav; Park, Sangwoo et al. (2018) Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase. J Biol Chem 293:191-202
Singh, Digvijay; Mallon, John; Poddar, Anustup et al. (2018) Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a). Proc Natl Acad Sci U S A 115:5444-5449
Panja, Subrata; Hua, Boyang; Zegarra, Diego et al. (2017) Metals induce transient folding and activation of the twister ribozyme. Nat Chem Biol 13:1109-1114
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
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
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
Ha, Taekjip (2016) Probing Nature's Nanomachines One Molecule at a Time. Biophys J 110:1004-7
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
Lee, Hye-Ra; Mitra, Jaba; Lee, Stacy et al. (2016) Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the cyclin D1 Gene. J Virol 90:1139-43

Showing the most recent 10 out of 127 publications