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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065367-02
Application #
6623172
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Lewis, Catherine D
Project Start
2002-04-01
Project End
2007-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
2
Fiscal Year
2003
Total Cost
$282,109
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Physics
Type
Schools of Engineering
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
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
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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
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Ha, Taekjip (2016) Probing Nature's Nanomachines One Molecule at a Time. Biophys J 110:1004-7

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