Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA.

Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (∼350 nucleotides per second) in the 5'→3' direction over relatively long distances (∼29,500 nucleotides). Surprisingly, we show the ssDNA-binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA.

Dynamics of DNA unwinding by helicases with frequent backward steps.

XPD (Xeroderma pigmentosum complementation group D) is a prototypical 5' - 3' translocating DNA helicase that exhibits frequent backward steps during DNA unwinding. Here, we propose a model of DNA unwinding by XPD. With the model we explain why XPD exhibits frequent backsteps while other helicases show rare backsteps. We explain quantitatively the single-molecule data on probability of -1-bp step and mean dwell time of one step versus ATP concentration for XPD at fixed large external force applied to the ends of the DNA hairpin to unzip the hairpin. We study DNA unwinding velocity, probability of -1-bp step and mean dwell time of one step for XPD versus external force at various ATP concentrations. We compare DNA unwinding dynamics of the 5' - 3' helicase XPD with that of 3' - 5' helicase RecQ. Our results show that the DNA unwinding velocity of XPD is sensitively dependent on the external force, which is contrast to RecQ that shows insensitive dependence of DNA unwinding velocity on the external force, explaining the experimental data showing that RecQ is an "optimally active" helicase while XPD is a "partially active" helicase. The DNA unwinding dynamics of different helicases under the external force is also studied.

Dynamics of monomeric and hexameric helicases.

Helicases are a ubiquitous class of enzymes that use the energy of ATP hydrolysis to unwind nucleic acid (NA) duplex. According to the structures, helicases can be classified as the non-ring-shaped (or monomeric) and ring-shaped (or hexameric). To understand the NA unwinding mechanism, here we study theoretically the unwinding dynamics of both the monomeric and hexameric helicases based on our proposed model. Various available single-molecule experimental data on unwinding speed of both the monomeric and hexameric helicases versus the external force applied to the ends of the NA duplex to unzip the duplex or versus the stability of the NA duplex are consistently and quantitatively explained. We provide quantitative explanations of the experimental data showing that while the unwinding speeds of some monomeric helicases are insensitively dependent on the external force they are sensitively dependent on the stability of the NA duplex. The experimental data showing that wild-type Rep translocates along ssDNA with a lower speed than RepΔ2B (removal of the 2B subdomain of Rep) and that RepΔ2B monomer can unwind DNA whereas the wild-type monomer is unable to unwind DNA are also quantitatively explained. Our studies indicate that although the monomeric and hexameric helicases show very different features on the dependence of NA unwinding speed upon the external force, they use much similar active mechanisms to unwind NA duplex.