DNA helicases are ATP-dependent motor proteins that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for replication, recombination and repair in all organisms. The principal investigator and his group propose to continue studies of two E. coli DNA helicases, Rep and UvrD (Helicase II), both of which appear to function as homo-dimers and function in replication and repair, respectively. The overall goal is to obtain a molecular understanding of the mechanism(s) by which these DNA helicases unwind duplex DNA and translocate along DNA and how these processes are coupled to ATP binding and hydrolysis. Quantitative biochemical and biophysical approaches will be used to examine the equilibria and kinetics of the interactions that are functionally important for DNA unwinding, such as DNA and nucleotide binding, ATP hydrolysis and protein self-assembly. This requires investigators to understand the molecular details of the known allosteric interactions that are key to the function of these multisubunit enzymes. The principal investigator and his group have proposed a """"""""subunit switching"""""""" model for how the Rep dimer translocates and unwinds duplex DNA that makes a number of testable predictions; many of the proposed studies are focused on testing this and other models. Dr. Lohman and his group will use transient kinetic approaches (stopped-flow fluorescence and chemical quenched-flow) to examine the pre-steady state kinetics and mechanism of ATP binding and hydrolysis by Rep and UvrD dimers in various DNA ligation states, some of which are proposed intermediates in DNA unwinding reactions. The thermodynamics, kinetics and mechanism of DNA binding will also be studied. In parallel, they will examine Rep and UvrD catalyzed unwinding of synthetic DNA substrates with the goal of developing a full kinetic mechanism for unwinding of synthetic DNA substrates with goal of developing a full kinetic mechanism for unwinding. Their recent x-ray crystal structure of Rep-ssDNA complexes (in collaboration with G. Waksman) has provided important structural insight and will aid the design of Rep and UrvD mutants to test the functional importance of different domains of the proteins for DNA and ATP binding, ATP hydrolysis, dimerization and DNA unwinding.
| Lohman, Timothy M; Fazio, Nicole T (2018) How Does a Helicase Unwind DNA? Insights from RecBCD Helicase. Bioessays 40:e1800009 |
| Ordabayev, Yerdos A; Nguyen, Binh; Niedziela-Majka, Anita et al. (2018) Regulation of UvrD Helicase Activity by MutL. J Mol Biol 430:4260-4274 |
| Nguyen, Binh; Ordabayev, Yerdos; Sokoloski, Joshua E et al. (2017) Large domain movements upon UvrD dimerization and helicase activation. Proc Natl Acad Sci U S A 114:12178-12183 |
| Tomko, Eric J; Lohman, Timothy M (2017) Modulation of Escherichia coli UvrD Single-Stranded DNA Translocation by DNA Base Composition. Biophys J 113:1405-1415 |
| Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto et al. (2016) Chemo-mechanical pushing of proteins along single-stranded DNA. Proc Natl Acad Sci U S A 113:6194-9 |
| Simon, Michael J; Sokoloski, Joshua E; Hao, Linxuan et al. (2016) Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation. J Mol Biol 428:2997-3012 |
| Petrova, Vessela; Chen, Stefanie H; Molzberger, Eileen T et al. (2015) Active displacement of RecA filaments by UvrD translocase activity. Nucleic Acids Res 43:4133-49 |
| 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 |
| Lee, Kyung Suk; Balci, Hamza; Jia, Haifeng et al. (2013) Direct imaging of single UvrD helicase dynamics on long single-stranded DNA. Nat Commun 4:1878 |
| Xie, Fuqian; Wu, Colin G; Weiland, Elizabeth et al. (2013) Asymmetric regulation of bipolar single-stranded DNA translocation by the two motors within Escherichia coli RecBCD helicase. J Biol Chem 288:1055-64 |
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