Rapid purification of helicase proteins and in vitro analysis of helicase activity

Tahmaseb, Kambiz; Matson, Steven W.

doi:10.1016/j.ymeth.2010.02.008

Cited by 4 publications

(3 citation statements)

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“…The uvrD303 allele was constructed in an expression plasmid as described under "Materials and Methods" and the protein was purified to apparent homogeneity using a rapid two-step purification procedure (29). To confirm the results presented previously (27), unwinding assays were performed using partial duplex substrates under multiple turnover conditions.…”

Section: Resultsmentioning

confidence: 95%

“…Using this cloning technique, the SmaI site in pTYB4-His is destroyed, but will ensure that only a Cterminal glycine will be added to the protein after purification, yielding essentially native protein. This construct enabled the rapid purification of the helicase as previously described (29). The uvrD303 allele was constructed using site-directed mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

“…The cells were stored at Ϫ80°C until use. The cells were lysed and the protein was purified as previously described using a combination of Talon (Clontech) and Chitin (New England Biolabs) resins to take advantage of the two affinity tags present on the overexpressed fusion protein (29). Protein that eluted from the Chitin column was dialyzed against UvrD storage buffer (31) and stored at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The UvrD303 Hyper-helicase Exhibits Increased Processivity

Meiners¹,

Tahmaseb²,

Matson

2014

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The UvrD303 Hyper-helicase Exhibits Increased Processivity

Meiners¹,

Tahmaseb²,

Matson

2014

Journal of Biological Chemistry

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Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA

Satusky

Johnson

Erie

2023

Biophysical Journal

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Special Methods collection on DNA helicases

Brosh

2010

Methods

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In this special Methods collection on DNA helicases, I have solicited articles from leading experts in the field with a priority to gather a defined series of papers on highly relevant topics that encompass biological, biochemical, and biophysical aspects of helicase function. The experimental approaches described provide an opportunity for both new and more experienced scientists to use the information for the design of their own investigations. The reader will find detailed methods for single-molecule studies, novel biochemical experiments, genetic analyses, and cell biological assays in a variety of systems with an emphasis placed on state-of-the-art techniques to measure helicase function. Contributing authors were strongly encouraged to provide a carefully constructed description of the methods employed so that others might use this information in a manner that will be useful for their own particular application and helicase of interest. This special issue of Methods dedicated to DNA helicases offers readers a treasure chest of unique experimental approaches and protocols focused on rapidly developing techniques that are useful for studying both in vivo and in vitro aspects of helicase function.DNA helicases have essential roles in virtually all aspects of nucleic acid metabolism involving the transient unwinding of the DNA double helix or other forms of DNA secondary structure. This special issue of Methods is comprised of a series of review articles by leaders in the field who deliver experimental procedures to study the molecular and cellular functions of DNA helicases and underlying mechanisms. We begin the special Methods collection with several distinct approaches and objectives for single-molecule studies of helicase proteins. This relatively new form of experimentation provides exciting approaches to derive insights into molecular aspects of helicase mechanism. The Seidel laboratory contributes a review article describing magnetic tweezer setups to study helicasecatalyzed DNA unwinding for different types of substrates and reaction conditions [1]. Techniques to modify the magnetic tweezer assay to acquire kinetic and thermodynamic information are also provided in the review by Kemmerich et al. [1]. The Spies laboratory has focused their review article on single-molecule sorting analyses that can be used to simultaneously quantify and distinguish the modulation of helicase-associated activities dependent on the post-translational modification of the helicase protein [2]. This paper by Bain et al. [2] provides detailed methodology to perform single-molecule sorting. The Kovacs laboratory delivers a review article for the Methods special collection that describes experimental approaches for highly mechanistic characterization of helicases that involves interactions with the nucleic acid, the nucleoside triphosphate hydrolysis cycle, and mechanochemical coupling of the two processes critical for duplex unwinding [3]. The experimental and theoretical aspects of fluorescence-based single-molecule ...

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Rapid purification of helicase proteins and in vitro analysis of helicase activity

Cited by 4 publications

References 46 publications

The UvrD303 Hyper-helicase Exhibits Increased Processivity

The UvrD303 Hyper-helicase Exhibits Increased Processivity

Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA

Special Methods collection on DNA helicases

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