Abstract
Although a number of eukaryotic DNA helicases have been identified biochemically and still more have been inferred from the amino acid sequences of the products of cloned genes, none of the cellular helicases or putative helicases has to date been implicated in eukaryotic chromosomal DNA replication. By the same token, numerous eukaryotic replication proteins have been identified, but none of these is a helicase. We have recently identified and characterized a temperature-sensitive yeast mutant, dna2ts, defective in DNA replication, and have cloned the corresponding gene (Kuo, C.-L., Huang, C,-H., and Campbell, J. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 30, 6465-6469; Budd, M. E., and Campbell, J. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7642-7646). The DNA2 gene is essential and encodes a 172-kDa protein with DNA helicase motifs in its C-terminal half and an N-terminal half with no similarity to any previously described protein (Budd, M. E., and Campbell, J. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7642-7646). Here we show that the helicase domain is required in vivo and that a 3' to 5' DNA helicase activity specific for forked substrates is intrinsic to the Dna2p. The N terminus is also essential for DNA replication. Thus, the structure of this new helicase is different from all previously characterized replicative helicases, which is consistent with the complex organization of eukaryotic replication forks, where the activities of not one but three essential DNA polymerases must be coordinated.
Highlights
We characterized a gene, DNA2, which complements a temperature-sensitive yeast strain defective in the elongation stage of DNA replication [1, 2]
The immunoaffinity-purified protein was shown to be associated with a DNA-dependent ATPase and a DNA helicase
The helicase is active only on a substrate with a forked structure, as is true of many prokaryotic and viral replicative helicases and appears to translocate in the 3Ј to 5Ј direction, the polarity of the leading strand at a replication fork [2]. While these experiments suggest that Dna2p is a replicative helicase, they are preliminary in two ways
Summary
We characterized a gene, DNA2, which complements a temperature-sensitive yeast strain defective in the elongation stage of DNA replication [1, 2]. An example of a DNA-dependent ATPase and helicase whose essential function may not require the helicase activity of the protein is the Rad3 protein. The ability of wild-type and ATPase mutant genes to complement the dna2-1ts strain and a dna2⌬ deletion strain was assessed.
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