RecA plays central roles in the homologous recombination to repair double-stranded DNA break damage in E. coli. A previously identified recA strain surviving high doses of UV radiation includes a dominant RecA E38K mutation. Using single-molecule experiments, we showed that the RecA E38K variant protein assembles nucleoprotein filaments more rapidly than the wild-type RecA. We also used a single-molecule fluorescence resonance energy transfer (smFRET) experiment to compare the nucleation cluster dynamics of wild-type RecA and RecA E38K mutants on various short ssDNA substrates. At shorter ssDNA, nucleation clusters of RecA E38K form dynamically, while only few were seen in wild-type RecA. RecA E38K also forms stable nuclei by specifically lowering the dissociation rate constant, k d. These observations provide evidence that greater nuclei stability and higher ssDNA binding affinity contribute to the observed enhanced recombination activity of the RecA E38K mutant. Given that assembly of RecA nucleoprotein filaments is the first committed step in recombinational repair processes, enhancement at this step gives rise to a more efficient recombinase.
E. coli RecBCD initiates homologous repair as well as degrades foreign DNA. Recognition of chi sequence (5'-GCTGGTGG-3') switches RecBCD from a destructive, nucleolytic mode into a repair-active one that promotes RecA-mediated recombination. RecBCD includes a 3'-to-5' single-stranded DNA (ssDNA) translocase in RecB subunit, a 5'-to-3' translocase in RecD, and a secondary translocase activity associated with RecBC. To understand how chi specifically affects each translocase activity, we directly visualized individual RecBCD translocating along DNA substrates containing a ssDNA gap of different polarities, with or without chi. Disappearance of RecBCD from the ssDNA signals the loss of the ssDNA translocase activity. For substrates containing a ssDNA gap that RecBCD encounters in the 3'-to-5' polarity (3'-to-5' ssDNA), wild-type RecBCD disappears from the DNA substrates with similarly high percentage, either with chi or without. This suggests that (1) the 3'-to-5' translocase in RecB is unaffected by chi and (2) it is low in processivity. With substrates containing a ssDNA gap that RecBCD encounters in the 5'-to-3' polarity (5'-to-3' ssDNA), we found that the leaving percentage increases significantly with chi, implying inactivation of the 5'-to-3' translocase of RecD upon chi recognition. Surprisingly, the RecD defective mutant RecBCD showed only ≈50 % leaving on 5'-to-3' ssDNA, directly revealing the presence of RecBC secondary translocase and its activity is unaffected by chi. Multiple ssDNA translocases within the RecBCD complex both before and after chi ensures processive unwinding of DNA substrates required for efficient recombination events.
Myosin VI physiologically functions as transporter and anchor in a cell. Recent single molecule high accuracy nano imaging (Nishikawa et al., 2010) revealed myosin VI has inchworm-like steps and hand-over-hand steps. These diverse stepping mode may relate to the switch between transporter and anchor, however, there's no evidence for that. Here, we examined the relashionship by a direct observation of stepping dynamics under tension, because tension triggers the switching between transporter and anchor. We achieved FIONA under tension by constructing nano sized spring using DNA nanotechnology (DNA origami). We found, during anchoring, myosin VI does not only prolong dwell time but repeats inchworm-like step and back steps to keep the anchoring state. 2P164 微生物の運動超分子マシナリーの単位ステップの直接観察: 滑走バクテリアと遊泳古細菌について Direct observation of unitary steps of supermolecular motility machineries of microorganisms: gliding bacterium and swimming archaeonMycoplasma mobile has a huge gliding machinery and glides on the substrate at the speed of 2.5 μm/sec. We proposed the "centipede model" in which leg protein portion of the machinery attaches to and detaches from sialylated oligosaccharides with a force production driven by ATP.Recently, we've detected this motion and reported M. mobile took 70 nm steps.Haloferax volcanii swims with a speed of 2 μm/sec by rotating archaeal flagella. It was functionally similar to a bacterial flagella though structurally resembled a bacterial typeIV pili. To clarify the force generation mechanism of flagella, we constructed a tethered cell assay. Notably, 6-9 steps per revolution were detected, which were roughly consistent with the periodicity of the active ATPase, the FlaI protein. 2P165好熱菌 F 1 のカップリングスキーム F 1 is an ATP-driven rotary molecular motor in which three catalytic sites, primarily hosted by a β subunit, hydrolyze ATP sequentially to power the rotation of γ subunit. The rotation occurs in steps of 120° per ATP, and the 120° step is further resolved into 80-90° and 40-30° substeps. In the basic coupling scheme, ATP binding starts rotation at 0°, and the ATP is hydrolyzed at 200°, and the ADP is released around 240° after a third ATP is bound. The timing of Pi release is yet unsettled: either at 200° or 320°. The timing of ATP hydrolysis, too, is not yet unequivocal. To complete the scheme, we observed the rotation driven by fluorescently (Cy3) labeled ATPγS and AMPPNP with single fluorophore imaging and by ATPγS with high speed imaging. 2P166高速 AFM によるミオシンⅥの機能の直接観察 Direct observation of functioning myosin VI by high-speedAFM Myosin VI, the only class of myosin that moves towards the minus end of actin filaments, performs various cellular functions including intracellular transports and structural maintenance of Golgi apparatus and stereocilla.Previous single molecule studies have demonstrated that myosin VI dimer moves processively with a step size (~36 nm) larger than that expected from its canonical lever-arm length. To gain an insight into the motile mechanism, we here use high-speed AFM t...
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