Abstract
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.
Highlights
DNA helicases are an abundant class of DNA metabolic enzymes, surpassing even the DNA polymerases in number and complexity, as well as in their resistance to experimental efforts to elucidate their functions
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
human DNA helicase B (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
Summary
Cloning and Sequencing of Human DNA Helicase B cDNA—A blast search of the Expressed Sequence Tag (EST) database (National Center for Biotechnology Information) revealed human sequences AA070301 (395 bp), AA256818 (311 bp), and AA256278 (258 bp) with significant. A recombinant baculovirus encoding human FEN-1 was constructed by inserting an NcoI/SspI fragment containing the FEN-1 cDNA into pVL1393 cut with NcoI and StuI, followed by co-transfection of the resulting pVL1393-FEN-1 construct with Baculogold DNA into Sf9 cells, as described above for the HDHB baculovirus. DNA-dependent ATPase Assay—The standard reaction mixture (10 l) contained 20 mM Tris-HCl (pH 7.5), 0.1 g/ml BSA, 0.5 mM DTT, 10 mM MgCl2, 50 M [␥32P]ATP (1 Ci/mmol) (Amersham Biosciences), varying amounts of DNA and RNA, and protein to be tested. Immunoprecipitation—Extracts from insect cells infected with baculoviruses expressing tagged HDHB, pol-prim, or FEN-1 were incubated for 1 h at 4 °C on a rotating wheel with agarose beads coupled to T7 tag antibody (Novagen, Madison, WI), collected by centrifugation, and washed twice with phosphate-buffered saline. Samples were dried and mounted on glass slides with 15 l of Pro-Long Antifade mounting medium (Molecular Probes, Eugene, OR)
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