Mechanism of DNA translocation in a replicative hexameric helicase

W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Nature (Impact Factor: 41.46). 08/2006; 442(7100):270-5. DOI: 10.1038/nature04943
Source: PubMed


The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.

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Available from: Leemor Joshua-Tor, Aug 14, 2014
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    • "The structures of homo-hexameric helicases E1 and Rho co-crystallized with their single- 361 stranded nucleic acid substrates strongly support a translocation mechanism where five motor 362 subunits contact five consecutive phosphates on the ssDNA/ssRNA backbone (Enemark and 363 Joshua-Tor, 2006; Thomsen and Berger, 2009), and the hydrolysis of one ATP is coupled to the 364 translocation of one nucleotide. Although an E1/Rho-like mechanism could potentially 365 rationalize how SpoIIIE easily traverses a dsMeP insert of 4 bp but not 5 bp (Figure 2), such a 366 model predicts that the motor would fail to traverse a stepping stone construct with a 1-bp MeP 367 probe (Figure 5b v). "
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    ABSTRACT: SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in B. subtilis. Here we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base-pair, we deduce that SpoIIIE's step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism that is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell.
    eLife Sciences 10/2015; 4. DOI:10.7554/eLife.09224 · 9.32 Impact Factor
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    • "Thus, T7 helicase and T7 DNAP respond differently to increasing GC content, which indicates that their DNA-unwinding mechanisms are fundamentally different. T7 helicase moves on DNA through sequential nucleotide hydrolysis and translocation mechanism, where each subunit of the ring takes turn in binding the incoming nucleotide (Liao et al., 2005; Crampton et al., 2006; Enemark and Joshua-Tor, 2006; Thomsen and Berger, 2009; Patel et al., 2011). Therefore at any given time, only the leading subunit of T7 helicase binds an incoming dTTP and reels in the nucleotide base from the fork junction (Sun et al., 2011). "
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    ABSTRACT: Leading strand DNA synthesis requires functional coupling between replicative helicase and DNA polymerase (DNAP) enzymes, but the structural and mechanistic basis of coupling is poorly understood. This study defines the precise positions of T7 helicase and T7 DNAP at the replication fork junction with single-base resolution to create a structural model that explains the mutual stimulation of activities. Our 2-aminopurine studies show that helicase and polymerase both participate in DNA melting, but each enzyme melts the junction base-pair partially. When combined, the junction base-pair is melted cooperatively provided the helicase is located one nucleotide ahead of the primer-end. The synergistic shift in equilibrium of junction base-pair melting by combined enzymes explains the cooperativity, wherein helicase stimulates the polymerase by promoting dNTP binding (decreasing dNTP Km), polymerase stimulates the helicase by increasing the unwinding rate-constant (kcat), consequently the combined enzymes unwind DNA with kinetic parameters resembling enzymes translocating on single-stranded DNA.
    eLife Sciences 05/2015; 4. DOI:10.7554/eLife.06562 · 9.32 Impact Factor
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    • "(A) In revolution motors, the right-handed DNA revolves within a left-handed channel, such as in the connector channels of bacteriophage Phi29 [46], P22 [45], and SPP1 [65]. (B) In rotation motors, the right-handed DNA rotates through a right-handed channel via the parallel thread, with RecA [76], DnaB [69] and E1 helicase [79] shown as examples. For E1 helicase, only the inside right-handed hairpin staircases that traces along the ssDNA are shown. "
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    ABSTRACT: Background Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days. Results Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is aligned with the channel could occur due to the conformational change of the phage motor channels from a left-handed configuration for DNA entry to a right-handed configuration for DNA ejection for host cell infection. Conclusions The revolution motor is widespread among biological systems, and can be distinguished from rotation motors by channel size and chirality. The revolution mechanism renders dsDNA void of coiling and torque during translocation of the lengthy helical chromosome, thus resulting in more efficient motor energy conversion.
    Cell and Bioscience 06/2014; 4(1):30. DOI:10.1186/2045-3701-4-30 · 3.63 Impact Factor
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