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18 DNA Helicases

James A. Borowiec


DNA helicases are proteins that use the energy of NTP hydrolysis to processively denature duplex DNA.1 As such, helicases can be considered similar to other mechanochemical motors that can catalyze directional movement along a polymeric lattice (e.g., microtubule-based motors as kinesin). Acting during DNA replication, helicases convert the duplex DNA to single strands and thereby activate the DNA for nascent strand synthesis by the replicative DNA polymerases. Because of the fundamental nature of this process, helicases also play essential roles during DNA repair and recombination as well as RNA transcription and are found in virtually all cells (Thömmes and Hübscher 1992; Lohman 1993; Matson et al. 1994).

Our knowledge of the DNA helicases acting during eukaryotic DNA replication is somewhat meager. The replicative helicases functioning during chromosomal replication for any eukaryote are unknown. However, information is available from the study of animal viruses that encode replicative helicases, as well as from prokaryotic replication systems. Here I outline the current understanding of DNA helicases that function in eukaryotic DNA replication.

DNA helicases are often first detected by a DNA-dependent or DNA-stimulated NTPase activity. Verification of DNA helicase activity is then accomplished using an assay that reveals the conversion of double-stranded DNA (dsDNA) into two single strands. The most common assay measures the release of a 32P-labeled oligonucleotide annealed to a larger ssDNA molecule, such as φX174 or M13 viral DNA (Fig. 1A) (Venkatesan et al. 1982; Matson et al. 1983). The radioactive substrate and product molecules differ greatly...

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