A Dominant-negative Mutant of Human DNA Helicase B Blocks the Onset of Chromosomal DNA Replication

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 singlestranded DNA, displayed robust 5-3 DNA helicase activity, and interacted physically and functionally with DNA polymerase ␣-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 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.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. Although prokaryotic and viral DNA helicases are comparatively well studied, eukaryotic DNA helicases remain poorly understood. The 134 helicaserelated genes encoded by Saccharomyces cerevisiae constitute more than 2% of the genome, but physiological functions of few of them are known (1). A better understanding of DNA replication, repair, and recombination pathways and the interplay among them in eukaryotic cells will depend on elucidation of the DNA helicases involved and their roles in each pathway.SV40 T antigen, a multifunctional viral protein, has served as a paradigm for a replicative helicase in eukaryotes (2, 3). It assembles on the viral origin of DNA replication, unwinds the parental strands, and directs the assembly of the cellular DNA polymerase ␣-primase (pol-prim) 1 (4) and replication protein A (RPA) (5) on the DNA, mediating the synthesis of the first RNA primers. A cellular DNA helicase, mouse DNA helicase B, was reported to share with T antigen the capacity to load pol-prim on RPA-coated single-stranded DNA and activate RNA primer synthesis (6, 7). Moreover, in a mutant derivative of FM3A mouse mammary carcinoma cells that express a thermolabile mutant of murine DNA helicase B, the onset of DNA replication was blocked at the non-permissive temperature (8), consistent with a possible role of the helicase in initiation of DNA replication. A cDNA encoding mouse DNA helicase B was recently cloned and characterized as a member of helicase superfamily 1 (9), which includes several well studied prokaryotic helicases, e.g. Escherichia coli uvrD/Helicase II, rep, recB(CD), and Bac...

DNA Helicase Activity of the RecD Protein from Deinococcus radiodurans

“…Others (Rrm3 and its close relative Pif1 from yeast, and proteins related to the TraA conjugative transfer protein of the Ti plasmid in Agrobacterium tumefasciens (48)) are helicases that are more distantly related to the RecD proteins and are also included in COG0507. The weak similarity of bacteriophage T4 Dda helicase and of human helicase B to the RecD-like proteins has been noted by others (49,50).…”

“…Dda may enable replication fork movement past bound proteins (56) and may also be involved in initiation of bacteriophage T4 replication (57). Helicase B is also thought to be involved in DNA replication, although its precise role is unknown (50). The conjugative transfer proteins (e.g.…”

Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus

“…The MCM helicase is a heterohexameric complex made up from Mcm 2±7 proteins, and is thought to be the eukaryotic replicative helicase (10), although it may not be the only helicase required for DNA replication initiation (11). The six Mcm subunits are highly related to each other, although each can be distinctly identi®ed as belonging to a certain subfamily, and these sub-classes are themselves conserved across eukaryotes.…”

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

“…SF1B helicases have clear roles in genome maintenance, although members of this family have not been nearly as well studied in vitro or in vivo as their SF1A helicase counterparts. Examples of the SF1B helicases include bacteriophage T4 Dda (DNA-dependent ATPase), a DNA replication enzyme; E. coli RecD, a critical component of the RecBCD helicase-nuclease required for end processing during double-strand break repair; and human DNA helicase B (HELB/hDHB), which is recruited into repair foci and enriched on chromatin following challenge with agents that cause replication stress (14,17,18). From these studies, it is clear that SF1B helicases are important for replication and repair; however, many bacterial SF1B helicases have not been studied, and their roles in genome maintenance remain unknown.…”