DNA structure specificity conferred on a replicative helicase by its loader

Abstract

Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.

FIGURE 1.

Stimulation of DnaB by excess DnaC depends on the structure of the DNA along which DnaB is translocating. Substrates 1 and 2 (A and B, respectively) were incubated with 150 nm DnaB and 0, 75, 150, 300, 600, or 1200 nm DnaC (lanes 2–7) for 4 min at 30 °C. Lane 1 contained neither DnaB nor C whereas lane 8 contained 1200 nm DnaC only. Shaded circles represent positions of the 5′ ³²P label. C, models of DnaB translocation along substrates 1 and 2. D, quantification of the extent of unwinding by DnaB of substrates 1 and 2 at increasing DnaC:B ratios. Error bars represent S.D.

FIGURE 2.

Basis of the DNA structure specificity of the inhibition of DnaB by excess DnaC. A–D, the rates of unwinding of substrates 1, 2, 5, and 4 by 150 nm DnaB at the indicated ratios of DnaC to DnaB. Error bars represent S.D.

FIGURE 3.

Prevention of DnaB translocation over dsDNA ameliorates the effects of excess DnaC on DnaB helicase activity. A and B, rates of unwinding of substrate 6 by 150 nm DnaB at the indicated ratios of DnaC to DnaB in the absence and the presence of streptavidin. C and D, rates of unwinding of substrate 2 in the absence and the presence of streptavidin. Conditions were otherwise identical to those used in A and B. Error bars represent S.D.

FIGURE 4.

Levels of unwinding of substrate 2 (A) and substrate 5 (B) by 150 nm DnaB in the presence of 0, 150, 300, and 600 nm DnaC as indicated. Reactions were performed for 4 min at 30 °C. Identities and concentrations of nucleotides present in the reactions are indicated.

FIGURE 5.

Inhibition of DnaB-catalyzed unwinding of ssDNA tailed Holliday junctions by excess DnaC. A, time courses of unwinding of substrate 10 by 600 nm DnaB in the absence of DnaC (lanes 1–6), with 600 nm DnaB and DnaC (lanes 7–12) and with 600 nm DnaB plus 2400 nm DnaC (lanes 13–18). Samples were taken at 0, 1, 2, 4, 8, and 16 min, as indicated in each time course. Shaded circles indicate positions of the 5′ ³²P label. B, time courses of unwinding of substrate 11. Concentrations of DnaB and DnaC, and time points, are as described in A. C, quantification of total levels of unwinding of substrates 10 (i) and 11 (ii) as a function of time by DnaB only (black squares), equimolar DnaB and DnaC (open squares), and 4-fold excess of DnaC over DnaB (open circles). Concentrations of DnaB and DnaC are as described in A. D, schematic of DnaB-catalyzed unwinding of substrates 10 (i) and 11 (ii and iii).

FIGURE 6.

A potential mechanism to explain the effects of DnaC on DnaB translocation along single-stranded DNA (A), forked DNA (B), and partial duplex DNA (C). Note that relief of inhibition of DnaB upon hydrolysis of ATP bound to DnaC (steps 2 and 3, and 6 and 7) could occur because of reduced affinity of DnaC^ADP for DnaB-ssDNA and/or because of the ability of DnaB to translocate along ssDNA when bound by DnaC^ADP.