Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased approximately 10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.

Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair, and recombination1,2. Bacteriophage T7 gene product 4 is a model hexameric helicase which has been observed to utilize dTTP, but not ATP, to unwind dsDNA as it translocates from 5′ to 3′ along ssDNA2–6. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate1,7. Here we address this question using a single molecule approach to monitor helicase unwinding. We discovered that T7 helicase does in fact unwind dsDNA in the presence of ATP and the unwinding rate is even faster than that with dTTP. However unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behavior was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found T7 helicase binds and hydrolyzes ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective Vmax, KM, and concentrations. In contrast, processivity does not follow a simple competitive behavior and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.DOI: http://dx.doi.org/10.7554/eLife.06249.001