SpringerProtocols: Abstract: Purification Strategy for Recombinant Formsof the Human Mitochondrial DNA Helicase

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Purification Strategy for Recombinant Formsof the Human Mitochondrial DNA Helicase

By: Tawn D. Ziebarth², Laurie S. Kaguni²

Abstract

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In this chapter, we present a streamlined purification for the production of near-homogeneous and high yield recombinant forms of the human mitochondrial DNA helicase. Minimizing the number of steps and the time elapsed for purification of this enzyme facilitates studies of its structure and mechanism and allows elucidation of native features of both wild-type- and human disease-related forms.

Affiliation(s): (2) Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA

Book Title: Mitochondrial DNA: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 554 | Pub. Date: Sep-01-2009 | Page Range: 121-126 | DOI: 10.1007/978-1-59745-521-3_8

Subject: Cell Biology

Key Words: mitochondrial DNA replication - DNA helicase - human - DNA unwinding

References

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1.	Ziebarth TD, Farr CL, Kaguni LS. Modular architecture of the hexameric human mitochondrial DNA helicase. J Mol Biol 2007;367(5):1382–91.

2.	Caruthers JM, McKay DB. Helicase structure and mechanism. Curr Opin Struct Biol 2002;12(1):123–33.

3.	Walker JE, Saraste M, Runswick MJ, Gay NJ. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. Embo J 1982;1(8):945–51.

4.	Korhonen JA, Gaspari M, Falkenberg M. TWINKLE Has 5′ -> 3′ DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein. J Biol Chem 2003;278(49):48627–32.

5.	Spelbrink JN, Li FY, Tiranti V, et al. Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat Genet 2001;28(3):223–31.

6.	Tyynismaa H, Sembongi H, Bokori-Brown M, et al. Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number. Hum Mol Genet 2004;13(24):3219–27.

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