DNA Helicase 

 Transient production of single-stranded DNA from the double helix is necessary prior to several DNA metabolic reactions, such as DNA replication, DNA repair, recombination, and transcription. DNA helicases unwind duplex DNA into two single DNA strands, ie, they disrupt the hydrogen bonds of double helical DNA by successively translocating along DNA, using the energy from hydrolysis of nucleoside triphosphates (NTP, usually ATP). Since the NTP hydrolysis is coupled to DNA binding, all helicases have DNA-dependent NTPase activity. Due to the involvement of helicases in various cell functions, most organisms have more than 10 helicases, and indeed, at least 12 helicases have been identified in Escherichia coli since the discovery of helicase I in 1976 (1). During DNA replication, a specific class of helicase unwinds duplex DNA ahead of the replication fork, which is the so-called “replicative helicase” and indispensable for cell proliferation. In addition to multiple helicases in cells, bacteriophages and eukaryotic viruses have their own helicases, which function in their specific propagation processes.

 Unwinding of a DNA duplex by helicases is assayed independently of their particular DNAmetabolic function, using partial duplex DNA that carries a short DNA annealed to a longer single-stranded DNA. The unwinding activity is manifested by the release of the short DNA from the duplex, which is detected as a faster-migrating DNA band in gel electrophoresis. The unwinding reaction proceeds in a unique direction defined as either a 5′-3′ or 3′-5′polarity with respect to the strand of DNA on which the helicase is bound. The polarity used to classify the enzyme (Table 1) is determined by similar unwinding assays using two duplex DNA carrying either 5′ or 3′ extensions of single-stranded DNA (Fig. 1). 

Figure 1. Helicase assay and determination of the polarity by electrophoresis.

Table 1. DNA Helicases and Their Properties

^a Nd = not determined.

Comparison of the primary structures of helicases revealed the presence of conserved motifs, which define several classes of the helicase family (2). A subfamily of helicases carry seven conserved motifs, but other subfamilies, which include most hexameric helicases (see below), retain only subsets of the motifs. Among the motifs, two called “A” and “B” are commonly detected in all helicases and function as a nucleotide-binding site. These two motifs are necessary but not sufficient for helicase functions, since the B motif corresponds to the so-called DEAD or DExH box, which is also observed in several RNA-binding proteins. Therefore, many putative DNA or RNA helicases are proposed on the basis of just their predicted primary structures, without the identification of actual biochemical activities.

 Although the precise mechanisms of their unwinding action are not understood, their characteristic oligomeric states, mainly as dimer or hexamer, propose simple explanations for their biochemical reactions. The dimeric form, such as the E. coli Rep helicase, provides an unwinding mechanism promoted by a rolling or inchworm movement, using two DNA-protein contacts. In this case, two DNA-binding sites alternate binding of single-stranded and duplex DNA, catalyzing unwinding. In the case of hexameric helicases, such as DnaB, T7 phage gene4 protein, and SV40 virus large T Antigen, they have a common ring structure, with a central hole through which DNA passes, as analyzed by electron microscopy. It is still unclear how DNA passes through the hexamer and how the unwinding reaction occurs within the molecule. Interestingly, these hexameric helicases are often members of large functional protein complexes, as in the case of typical replicative helicases, and are expected to have roles as motor molecules that may simultaneously unwind the DNA and transfer the protein complexes along DNA.

 Replicative helicases have been identified only from bacteria, bacteriophage, and viruses, not from eukaryotic cells. This is mainly because there has not been any reliable in vitro replication system in eukaryotes to confirm whether a helicase of interest is functional in replication or not. A yeast essential gene, dna2, has been identified as a DNA replication mutant by screening using a permeabilized cell replication assay (3). Since this gene product has five conserved helicase motifs and actually exhibits helicase activity, it is a candidate for the yeast replicative helicase, although there is insufficient direct evidence. The known replicative helicases often have close connections with other replication activities and sometimes possess additional characteristics; eg, primase activity in T7 gene4 helicase and replication origin binding in SV40 T antigen. T antigen also interacts physically with several replication proteins and cooperatively promotes the initiation of viral DNA replication. The E. coli replicative helicase, DnaB, exhibits the closer physical and functional link with the replication fork components, and the rate of helicase movement is coordinately stimulated by interaction with the DNA polymerase (4).

Recent improvements in identifying the causative genes of many human heritable diseases revealed that the defects of certain helicase functions cause several human diseases, including Xeroderma pigmentosum, Cockayne's syndrome, Bloom's syndrome Werner's syndrome, and ATR-X syndrome (5) . These diseases are apparently related to the defects of genes whose functions are involved in nucleotide excision repair, recombination, replication fidelity, and chromosome transmission, and they exhibit cancer-prone phenotypes because of the difficulty in maintaining genome integrity. Such human disease-related helicases are conserved among eukaryotes, and their counterparts are often identified as RAD genes in yeast (Table 1).

References

1. M. Abdel-Monem et al. (1976) Eur. J. Biochem. 65, 441–449. 

2. A. E. Gorbalenya and E. V. Koonin (1993) Curr. Opin. Struct. Biol. 3, 419–429. 

3. M. E. Budd and J. L. Campbell (1995) Proc. Natl. Acad. Sci USA 92, 7642–7646.

4. S. Kim et al. (1996) Cell 84 643–650. 

5. T. M. Lohman and K. P. Bjornson (1996) Ann. Rev. Biochem. 65, 169–214.