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The Saccharomyces cerevisiae RAD3 gene, which is required for cell viability and excision repair of damaged DNA, encodes an 89-kDa protein that has a single-stranded DNA-dependent ATPase activity. We now show that the RAD3 protein also possesses a helicase activity that unwinds duplex regions in DNA substrates constructed by annealing DNA fragments of 71-851 nucleotides to circular, single-stranded M13 DNA. The DNA helicase activity is dependent on the hydrolysis of ATP, has a pH optimum of approximately 5.6, and is inhibited by antibodies raised against a truncated RAD3 protein produced in Escherichia coli. The RAD3 helicase translocates along single-stranded DNA in the 5'----3' direction. The direction of RAD3 helicase movement is consistent with the possibility that it unwinds DNA duplexes in advance of the replication fork during DNA replication.

RAD3 protein of Saccharomyces cerevisiae is a DNA helicase.