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



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 helicase-related genes encoded by Saccharomyces cerevisiae constitute more than 2% of the genome, but physiological functions of few of them are known. 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. 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) and replication protein A (RPA) 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. 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, 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, which includes several well studied prokaryotic helicases, e.g. Escherichia coli uvrD/Helicase II, rep, recB(CD), and Bacillus stearothermophilus PcrA. However, recombinant mouse DNA helicase B expressed from the murine cDNA has not yet been described.

To explore the role of a putative human ortholog of mouse DNA helicase B in human DNA replication, we have cloned a cDNA encoding human DNA helicase B (HDHB), expressed and purified the recombinant polypeptide, and characterized its activity in vitro and after microinjection into human cells. The results demonstrate that HDHB is closely related in sequence to the mouse helicase and is widely expressed in cells and tissues with an active DNA metabolism. The properties of recombinant HDHB confirm and extend those reported for the helicase B purified from mouse cells, strongly suggesting a role for HDHB in an aspect of DNA metabolism that depends on pol-prim priming activity.

HDHB Is the Human Ortholog of Mouse DNA Helicase B-- Comparison of the deduced amino acid sequence of HDHB cDNA with that of mouse DNA helicase B cDNA suggests that HDHB is closely related to DNA helicase B. Consistent with the sequence homology, preliminary characterization of the enzymatic properties of recombinant HDHB revealed similarities with DNA helicase B purified from mouse cells. Both proteins displayed robust ATPase activity dependent on single-stranded DNA, preferred ATP and dATP as substrates, and unwound several hundred base pairs of duplex DNA with 5'-3' polarity.

In addition to these similarities, analysis of helicase-deficient mutants of HDHB and mouse DNA helicase B suggests that both proteins function in a process needed for the onset of S phase. Introduction of a helicase-deficient HDHB protein into human cells in G1 inhibited the G1/S transition, suggesting a dominant-negative phenotype.




The sequence homology of DNA helicase B with prokaryotic recombination proteins recD and T4 dda provides a third possible clue to its functional role. If, like recD, DNA helicase B participates in initiating homologous DNA recombination during S phase, it might interact with pol-prim and RPA during reactivation of stalled or broken replication forks. However, if HDHB performs a recD-like function, one might expect it to associate with recBC-like proteins or with other nucleases. In summary, although the specific roles of HDHB in DNA metabolism remain to be elucidated, the work presented here and elsewhere offers some useful clues and a foundation for future studies toward this goal.

Taneja P, et. al. (2002). A Dominant-negative Mutant of Human DNA Helicase B Blocks the Onset of Chromosomal DNA Replication. < a href="http://www.jbc.org/cgi/content/full/277/43/40853">J. Biol. Chem., Vol. 277, Issue 43, 40853-40861.