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J. Biol. Chem., Vol. 280, Issue 46, 38117-38120, November 18, 2005

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The DEXH Protein Product of the DHX36 Gene Is the Major Source of Tetramolecular Quadruplex G4-DNA Resolving Activity in HeLa Cell Lysates^*

James P. Vaughn, Steven D. Creacy1, Eric D. Routh, Christi Joyner-Butt, G. Scott Jenkins, Sandra Pauli, Yoshikuni Nagamine, and Steven A. Akman2

From the Department of Cancer Biology and the Comprehensive Cancer Center of Wake Forest Medical Center, Winston-Salem, North Carolina 27157 and the Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, CH-4058 Basel, Switzerland

G4-DNA is a highly stable alternative DNA structure that can form spontaneously in guanine-rich regions of single-stranded DNA under physiological conditions. Since a number of biological processes create such single-stranded regions, G4-DNA occurrence must be regulated. To date, resolution of tetramolecular G4-DNA into single strands (G4-resolvase activity) has been observed only in recombinant RecQ DNA helicases. We previously reported that human cell lysates possess tetramolecular G4-DNA resolving activity (Harrington, C., Lan, Y., and Akman, S. (1997) J. Biol Chem. 272, 24631–24636). Here we report the first complete purification of a major non-RecQ, NTP-dependent G4-DNA resolving enzyme from human cell lysates. This enzyme is identified as the DEXH helicase product of gene DHX36 (also known as RHAU). G4-DNA resolving activity was captured from HeLa cell lysates on G4-DNA affinity beads and further purified by gel filtration chromatography. The DHX36 gene product was identified by mass spectrometric sequencing of a tryptic digest from the protein band on SDS-PAGE associated with activity. DHX36 was cloned within a His₆-tagging vector, expressed, and purified from Escherichia coli. Inhibition and substrate resolution assays showed that recombinant DHX36 protein displayed robust, highly specific G4-DNA resolving activity. Immunodepletion of HeLa lysates by a monoclonal antibody to the DHX36 product removed ca. 77% of the enzyme from lysates and reduced G4-DNA resolving activity to 46.0 ± 0.4% of control, demonstrating that DHX36 protein is responsible for the majority of tetramolecular G4-DNA resolvase activity.

Received for publication, August 11, 2005 , and in revised form, September 7, 2005.

^* This work was supported by Grant 2-P30 CA12197 of the Comprehensive Cancer Center of Wake Forest Medical Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data.

¹ S. D. C. is a primary author of this publication along with J. P. V. and S. A. A.

² To whom correspondence should be addressed: Dept. of Cancer Biology and the Comprehensive Cancer Center of Wake Forest Medical Center, Medical Center Boulevard, Winston-Salem, NC 27157. Tel.: 336-716-0230; Fax: 336-716-0255 E-mail: sakman{at}wfubmc.edu.

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