A dominant-negative mutant of human DNA helicase B blocks the onset of chromosomal DNA replication.
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 G(1), the mutant proteins inhibited DNA synthesis, whereas the wild type protein had no effect. Injection of wild type or mutant protein into cells at G(1)/S did not prevent DNA synthesis. The results suggest that HDHB function is required for S phase entry.
Department of Biological Sciences and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37232, USA.
Poonam
P
Taneja
Jinming
J
Gu
Rui
R
Peng
Ryan
R
Carrick
Fumiaki
F
Uchiumi
Robert D
RD
Ott
Eric
E
Gustafson
Vladimir N
VN
Podust
Ellen
E
Fanning
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017931:DNA Primers; D004261:DNA Replication / Q000502:physiology*; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008845:Microinjections; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D000251:Adenosine Triphosphatases; C484263:HELB protein, human; C484264:Helb protein, mouse; D004265:DNA Helicases