Cellular replisomes are powered by flex-fuel motors for unwinding DNA.

DNA replication relies on hexameric, ring-shaped helicases to unwind parental DNA for supporting fork progression over tens of thousands of base pairs. Oddly, biochemical studies have suggested that, on their own, replicative helicases are rather limited motors that struggle to couple rapid movement to nucleotide turnover (typically believed to be solely ATP). Here, single-molecule studies reveal that when properly loaded, the Escherichia coli (E. coli) replicative helicase, DnaB, is a tremendously fast single-stranded DNA translocase that moves up to three times more rapidly than the replisome (3 knt/s). Translocation is highly processive, resistant to pulling force/high salt, and can displace short, 3'-tailed DNA duplexes without apparent changes in speed. Surprisingly, we find that the loader for DnaB, DnaC, can use any rNTP or dATP for depositing DnaB onto ssDNA and that the helicase itself also must hydrolyze nucleotide for stable loading. DnaB translocation also turns out to be supported by any r/dNTP, a property shown to extend to the eukaryotic CMG replicative helicase. Overall, the DNA unwinding engines that support cellular replisomes are highly indiscriminate of their fuel source, a feature that may be of utility during times when cells encounter nucleotide pool stress but have committed to DNA synthesis.