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

ABSTRACT 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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    • "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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    • "We therefore built a comparative predicted model of RepC using the experimentally determined structure of its close homologue BPV-E1 as a template (Fig. 7). Although the amino acid sequences of the helicase domains of these two proteins are only ~16 % identical, many features of the predicted model of the resulting hexamers justifies our biochemical data and is comparable with other hexameric helicases (Li et al., 2003; Yoon-Robarts et al., 2004; Enemark & Joshua-Tor, 2006). For example, the model places the Walker A and Walker B motifs at the interface between subunits of the hexamer, suitable for ATP binding. "
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    ABSTRACT: Replication initiator protein (Rep) is indispensible for rolling-circle replication of geminiviruses, a group of plant infecting circular single stranded DNA (ssDNA) viruses. However, the mechanism of DNA unwinding by circular ssDNA virus encoded helicases is unknown. To understand geminivirus Rep function, we compared the sequence and secondary structure of Rep with those of bovine papillomavirus-E1 and employed charged residue-to-alanine scanning mutagenesis to generate a set of single-substitution mutants in Walker A (K227), in Walker B (D261, 262), and within or adjacent to the B' motif (K272, K286 and K289). All mutants were asymptomatic and viral accumulation could not be detected by Southern blot from both tomato and N. benthamiana plants. Furthermore, the K272 and K289 mutants were deficient in DNA binding and unwinding. Biochemical studies and modelling data based on comparison with the known structures of SF3 helicases suggest that the conserved lysine (K289) located in a predicted β-hairpin loop may interact with ssDNA, while lysine 272 in the B' motif (K272) located on the outer surface of the protein is presumably involved in coupling ATP-induced conformational changes to DNA binding. To the best of our knowledge, this is the first time that the roles of the B' motif and the adjacent β-hairpin loop in geminivirus replication have been elucidated.
    Journal of General Virology 04/2014; 95(Pt_7). DOI:10.1099/vir.0.064923-0 · 3.18 Impact Factor
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