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Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase

Abstract

Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid1,2,3, and are involved in several human diseases. Some function as hexameric rings4, but the functional form of non-hexameric helicases has been debated5,6,7,8,9,10. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays—which do not interfere with biological activity—to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions (‘stalls’) that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein–DNA interactions.

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Acknowledgements

We thank C. Chidsey for suggesting the use of a PEG surface, and T. Ho for synthesis and purification of the oligonucleotides. This work was supported by the NSF and AFOSR (S.C.), by the NIH (T.M.L.), and by a Searle scholars award, the NIH, an NSF CAREER award, the Research Corporation, and the UIUC research board (T.H.)

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Correspondence to Taekjip Ha.

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The authors declare that they have no competing financial interests.

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Further reading

Figure 1: FRET assay for single DNA unwinding.
Figure 2: ATP-dependent, junction-specific fluctuations of Rep monomer.
Figure 3: Mechanism of unwinding stall and re-initiation.

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