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J. Biol. Chem., Vol. 277, Issue 43, 40853-40861, October 25, 2002
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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 The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF319995.
A Dominant-negative Mutant of Human DNA Helicase B Blocks the
Onset of Chromosomal DNA Replication*
,
,
-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 -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.
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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