• Proceedings of the Microbiological Society of Korea Conference
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• 2008.05a
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• pp.121-123
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• 2008

• Journal of Life Science
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• v.14 no.1
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• pp.131-140
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• 2004

T7 bacteriophage gp4 is the replicative DNA helicase that unwinds double-stranded DNA by utilizing dTTP hydrolysis energy. The quaternary structure of the active form of T7 helicase is a hexameric ring with a central channel. Single-stranded DNA passes through the central channel of the hexameric ring as the helicase translocates $5'\rightarrow3'$ along the single-stranded DNA. The DNA unwinding was measured by rapid kinetic methods and showed a lag before the single-stranded DNA started to accumulate exponentially. This behavior was analyzed by a kinetic stepping model for the unwinding process. The observed lag phase increased as predicted by the model with increasing double-stranded DNA length. Trap DNA added in the reaction had no effect on the amplitudes of double-stranded DNA unwound, indicating that the $\tau7$ helicase is a highly processive helicase. Global fitting of the kinetic data to the stepping model provided a kinetic step size of 10-11 bp/step with a rate of $3.7 s^{-1}$ per step. Both the mechanism of DNA unwinding and dTTP hydrolysis and the coupling between the two are unaffected by temperature from $4∼37^{\circ}C$. Thus, the kinetic stepping for dsDNA unwinding is an inherent property of tile replicative DNA helicase.

• Bulletin of the Korean Chemical Society
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• v.27 no.10
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• pp.1618-1622
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• 2006

Bacteriophage T7 gp4A' is a ring-shaped hexameric DNA helicase that catalyzes duplex DNA unwinding using dTTP hydrolysis as an energy source. To investigate the effect of single-stranded DNA (ssDNA) on the kinetic pathway of dTTP hydrolysis by the T7 DNA helicase complexed with ssDNA, we have first determined optimal concentration of long circular M13 single-stranded DNA and pre-incubation time in the absence of $Mg^{2+}$ which is necessary for the helicase-ssDNA complex formation. Steady state dTTP hydrolysis in the absence of $Mg^{2+}$ by the helicase-ssDNA complex provided $k_{cat}$ of $8.5\;{\times}\;10^{-3}\;sec^{-1}$. Pre-steady state kinetics of the dTTP hydrolysis by the pre-assembled hexameric helicase was monitored by using the rapid chemical quench-flow technique both in the presence and absence of M13 ssDNA. Pre-steady state dTTP hydrolysis showed distinct burst kinetics in both cases, indicating that product release step is slower than dTTP hydrolysis step. Pre-steady state burst rates were similar both in the presence and absence of ssDNA, while steady state dTTP hydrolysis rate in the presence of ssDNA was much faster than in the absence of ssDNA. These results suggest that single-stranded DNA stimulates dTTP hydrolysis reaction by T7 helicase by enhancing the rate of product release step.

• Molecules and Cells
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• v.21 no.1
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• pp.129-134
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• 2006

Lon, also known as protease La, belongs to a class of ATP-dependent serine protease. It plays an essential role in degradation of abnormal proteins and of certain short-lived regulatory proteins, and is thought to possess a Ser-Lys catalytic dyad. To examine the structural organization of Lon, we performed an electron microscope analysis. The averaged images of Lon with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity. The side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since a Lon subunit possesses two large regions containing nucleotide binding and proteolytic domains, each layer of the Lon hexamer appears to consist of the side projections of one of the major domains arranged in a ring. Lon showed a strong tendency to form hexamers in the presence of $Mg^{2+}$, but dissociated into monomers and/or dimers in its absence. Moreover, $Mg^{2+}$-dependent hexamer formation was independent of ATP. These results indicate that Lon has a hexameric ring-shaped structure with a central cavity, and that the establishment of this configuration requires $Mg^{2+}$, but not ATP.

• Molecules and Cells
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• v.44 no.2
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• pp.79-87
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• 2021

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.