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Originally published In Press as doi:10.1074/jbc.M208067200 on August 13, 2002

J. Biol. Chem., Vol. 277, Issue 43, 40853-40861, October 25, 2002
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A Dominant-negative Mutant of Human DNA Helicase B Blocks the Onset of Chromosomal DNA Replication*

Poonam TanejaDagger , Jinming Gu, Rui Peng, Ryan Carrick§, Fumiaki Uchiumi, Robert D. Ott, Eric Gustafson||, Vladimir N. Podust, and Ellen Fanning**

From the Department of Biological Sciences and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37232

A cDNA encoding a human ortholog of mouse DNA helicase B, which may play a role in DNA replication, has been cloned and expressed as a recombinant protein. The predicted human DNA helicase B (HDHB) protein contains conserved helicase motifs (superfamily 1) that are strikingly similar to those of bacterial recD and T4 dda proteins. The HDHB gene is expressed at low levels in liver, spleen, kidney, and brain and at higher levels in testis and thymus. Purified recombinant HDHB hydrolyzed ATP and dATP in the presence of single-stranded DNA, displayed robust 5'-3' DNA helicase activity, and interacted physically and functionally with DNA polymerase alpha -primase. HDHB proteins with mutations in the Walker A or B motif lacked ATPase and helicase activity but retained the ability to interact with DNA polymerase alpha -primase, suggesting that the mutants might be dominant over endogenous HDHB in human cells. When purified HDHB protein was microinjected into the nucleus of cells in early G1, the mutant proteins inhibited DNA synthesis, whereas the wild type protein had no effect. Injection of wild type or mutant protein into cells at G1/S did not prevent DNA synthesis. The results suggest that HDHB function is required for S phase entry.

* This work was supported in part by National Institutes of Health Grant GM52948 (to E. F.), National Institutes of Health Grant CA68485 (to Vanderbilt-Ingram Cancer Center core facilities), by Vanderbilt University, and by a leave from the Science University of Tokyo (to F. U.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF319995.

Dagger Present address: Alpha Innotech Corp., 2401 Merced St., San Leandro, CA 94577.

§ Present address: Loyola University Chicago, Stritch School of Medicine, 2160 S. First Ave., Student Box 489, Maywood, IL 60153.

Present address: Science University of Tokyo, Pharmaceutical Sciences, 12 Ichigaya Funagawara-Machi, Shinjuku-ku, Tokyo, Japan.

|| Present address: Dept. of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912.

** To whom correspondence should be addressed: Dept. of Biological Sciences, VU Station B 351634, Vanderbilt University, Nashville, TN 37235-1634. Tel.: 615-343-5677; Fax: 615-343-6707; E-mail: fannine@ctrvax.vanderbilt.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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