Partial Duplex Substrates Research Articles

RecQ DNA Helicase of Escherichia coli: Characterization of the Helix-unwinding Activity with Emphasis on the Effect of Single-stranded DNA-binding Protein

RecQ protein of Escherichia coli is a DNA helicase implicated in the RecF pathway of genetic recombination. To gain insight into the mode of its action, the effect of single-stranded DNA-binding proteins (SSBs) on the RecQ-mediated unwinding reaction was investigated. When the unwinding of M13-based, circular partially duplex substrates was measured as a function of the enzyme dose, a markedly sigmoidal relation was revealed, with relatively large amounts of the enzyme being necessary for substantial unwinding to occur. For instance, unwinding 50% of a 71 base-pair (bp) partial duplex substrate in ten minutes required an enzyme-to-substrate molar ratio of about 60. However, these features, indicating the enzyme's "inefficiency", were reversed by SSBs: in the presence of a saturating amount of E. coli SSB the sigmoidal relation was converted to a typically hyperbolic one, and the enzyme-to-substrate molar ratio at 50% unwinding of the 71 bp substrate was reduced to as low as 0·5. Phage T4 gene 32 protein also showed similar stimulatory activity. Further, the single-stranded DNA-dependent ATPase activity of RecQ was found to be relatively insensitive to E. coli SSB; its large excess brought about only a 60% inhibition. It is postulated that RecQ helicase is highly adapted to an SSB-rich environment, where the strand exchange reaction mediated by RecA protein, perhaps coupled closely with the RecQ reaction, should also take place.

Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction.

Helicase I has been purified to greater than 95% homogeneity from an F+ strain of Escherichia coli, and characterized as a single-stranded DNA-dependent ATPase and a helicase. The duplex DNA unwinding reaction requires a region of ssDNA for enzyme binding and concomitant nucleoside 5'-triphosphate hydrolysis. All eight predominant nucleoside 5'-triphosphates can satisfy this requirement. Unwinding is unidirectional in the 5' to 3' direction. The length of duplex DNA unwound is independent of protein concentration suggesting that the unwinding reaction is highly processive. Kinetic analysis of the unwinding reaction indicates that the enzyme turns over very slowly from one DNA substrate molecule to another. The ATP hydrolysis reaction is continuous when circular partial duplex DNA substrates are used as DNA effectors. When linear partial duplex substrates are used ATP hydrolysis is barely detectable, although the kinetics of the unwinding reaction on linear partial duplex substrates are identical to those observed using a circular partial duplex DNA substrate. This suggests that ATP hydrolysis fuels continuous translocation of helicase I on circular single-stranded DNA while on linear single stranded DNA the enzyme translocates to the end of the DNA molecule where it must slowly dissociate from the substrate molecule and/or slowly associate with a new substrate molecule, thus resulting in a very low rate of ATP hydrolysis.