Biotin-Streptavidin-Labeled Oligonucleotides as Probes of Helicase Mechanisms

Morris, Patrick D.; Tackett, Alan J.; Raney, Kevin D.

doi:10.1006/meth.2000.1116

Cited by 31 publications

(31 citation statements)

References 28 publications

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“…Poly(U) was purchased from Amersham Biosciences. DNA oligonucleotides were from Integrated DNA Technologies and purified by preparative gel electrophoresis (38). [␥-…”

Section: Methodsmentioning

confidence: 99%

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Jennings

Mackintosh

Harrison

et al. 2009

Journal of Biological Chemistry

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Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nM. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.Helicases are ubiquitous enzymes required for virtually all cellular processes involving nucleic acids, including replication, transcription, translation, repair, and recombination (1-5). These enzymes catalyze unwinding of double-stranded DNA or RNA by converting chemical energy from ATP hydrolysis into mechanical energy for nucleic acid strand separation. However, there is considerable variability in the quaternary structure of the active forms of helicases. Some helicases function effectively in unwinding activities as monomers, whereas others are active as dimers or oligomers. For example, bacteriophage T4 gp41 helicase and Escherichia coli DnaB helicase form hexameric structures that encircle and sequester single-stranded DNA (6, 7). Indeed, a large number of helicases form and function as hexameric structures (3). PcrA, a Gram-positive bacterial helicase, translocates on single-stranded DNA as a monomer (8) and has been proposed to unwind double-stranded DNA as a monomer (9). Bacteriophage T4 Dda helicase has activity in the monomeric form (10) but demonstrates more efficient unwinding activity under conditions where multiple helicase molecules bind a given substrate molecule (11). It has been shown that E. coli Rep helicase unwinds DNA as a dimer (12). UvrD has...

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“…Poly(U) was purchased from Amersham Biosciences. DNA oligonucleotides were from Integrated DNA Technologies and purified by preparative gel electrophoresis (38). [␥-…”

Section: Methodsmentioning

confidence: 99%

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Jennings

Mackintosh

Harrison

et al. 2009

Journal of Biological Chemistry

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“…Using the stepping model used previously for helicases, 56,57 the initiation, translocation and TFO displacement events during translocation of a type I enzyme can be depicted using a simple lattice scheme (the assumptions necessary for this scheme are described in detail by Szczelkun 56 ). This scheme could be readily adapted for other roadblock assays, such as those involving biotin displacement: [45][46][47][48][49] …”

Section: Kinetic Models Of Triplex Displacementmentioning

confidence: 99%

“…[41][42][43][44] A second example of a roadblock assay involves the displacement of streptavidin from biotin at the 5 0 or 3 0 end of a synthetic oligonucleotide. 45 This has been used with a number of helicases to monitor directional bias and rates of ssDNA translocation. [46][47][48][49] This assay could be readily developed for measuring translocation on dsDNA (assuming that the translocating enzyme can displace the streptavidin) and would yield similar kinetics to the triplex assay.…”

Section: Introductionmentioning

confidence: 99%

Continuous Assays for DNA Translocation Using Fluorescent Triplex Dissociation: Application to Type I Restriction Endonucleases

McClelland

Dryden

Szczelkun

2005

Journal of Molecular Biology

108

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“…The free energy released due to the hydrolysis of one ATP molecule should be sufficient to separate several base pairs of the DNA duplex. On the other hand, disruption of the streptavidin-biotin interaction – a common reporter for translocase activity [16] – is energetically equivalent to multiple ATP molecules. Despite these facts, many helicases readily displace streptavidin from biotinylated DNA but cannot unwind duplex DNA.…”

Section: Introductionmentioning

confidence: 99%

Two steps forward, one step back: Determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers

Spies

2014

DNA Repair

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XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′-3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interaction within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein-DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).

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Biotin-Streptavidin-Labeled Oligonucleotides as Probes of Helicase Mechanisms

Cited by 31 publications

References 28 publications

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Continuous Assays for DNA Translocation Using Fluorescent Triplex Dissociation: Application to Type I Restriction Endonucleases

Two steps forward, one step back: Determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers

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