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Analysis of the HindIII-catalyzed reaction by time-resolved crystallography.

Kawamura T, Kobayashi T, Watanabe N - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites.DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion.In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

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Affiliation: Synchrotron Radiation Research Center, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan.

ABSTRACT
In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII-DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

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OMIT maps for detecting the cleavage of the phosphodiester bond in the (a) 0 s, (b) 25 s, (c) 40 s, (d) 60 s and (e) 230 s structures. HindIII and the DNA molecules are shown as stick models. Water molecules are shown as small cyan-coloured balls. Sodium ions in the 0 s structure and manganese ions in structures of the crystals cooled at the other time points are shown as large balls. Electron-density contours are the same as in Fig. 2 ▶. Part of the DNA structure of DA4–DA5 is omitted for the calculations.
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fig3: OMIT maps for detecting the cleavage of the phosphodiester bond in the (a) 0 s, (b) 25 s, (c) 40 s, (d) 60 s and (e) 230 s structures. HindIII and the DNA molecules are shown as stick models. Water molecules are shown as small cyan-coloured balls. Sodium ions in the 0 s structure and manganese ions in structures of the crystals cooled at the other time points are shown as large balls. Electron-density contours are the same as in Fig. 2 ▶. Part of the DNA structure of DA4–DA5 is omitted for the calculations.

Mentions: The scissile phosphodiester bond of the DNA backbone in the HindIII cognate sequence A/AGCTT was scrutinized using OMIT maps (Fig. 3 ▶). The DNA structure of DA4 and DA5 was omitted from the map calculations. The phosphodiester bond is cleaved at 230 s (Fig. 3 ▶e). At 60 s, however, the electron-density map indicates the presence of a significant amount of uncleaved dsDNA (Fig. 3 ▶d). In structures of the complex prior to 40 s there are no significant conformational changes in the structure of dsDNA. In the 230 s structure, conformational changes directly related to cleavage of the phosphodiester bond between DA4 and DA5 in the DNA are observed.


Analysis of the HindIII-catalyzed reaction by time-resolved crystallography.

Kawamura T, Kobayashi T, Watanabe N - Acta Crystallogr. D Biol. Crystallogr. (2015)

OMIT maps for detecting the cleavage of the phosphodiester bond in the (a) 0 s, (b) 25 s, (c) 40 s, (d) 60 s and (e) 230 s structures. HindIII and the DNA molecules are shown as stick models. Water molecules are shown as small cyan-coloured balls. Sodium ions in the 0 s structure and manganese ions in structures of the crystals cooled at the other time points are shown as large balls. Electron-density contours are the same as in Fig. 2 ▶. Part of the DNA structure of DA4–DA5 is omitted for the calculations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321485&req=5

fig3: OMIT maps for detecting the cleavage of the phosphodiester bond in the (a) 0 s, (b) 25 s, (c) 40 s, (d) 60 s and (e) 230 s structures. HindIII and the DNA molecules are shown as stick models. Water molecules are shown as small cyan-coloured balls. Sodium ions in the 0 s structure and manganese ions in structures of the crystals cooled at the other time points are shown as large balls. Electron-density contours are the same as in Fig. 2 ▶. Part of the DNA structure of DA4–DA5 is omitted for the calculations.
Mentions: The scissile phosphodiester bond of the DNA backbone in the HindIII cognate sequence A/AGCTT was scrutinized using OMIT maps (Fig. 3 ▶). The DNA structure of DA4 and DA5 was omitted from the map calculations. The phosphodiester bond is cleaved at 230 s (Fig. 3 ▶e). At 60 s, however, the electron-density map indicates the presence of a significant amount of uncleaved dsDNA (Fig. 3 ▶d). In structures of the complex prior to 40 s there are no significant conformational changes in the structure of dsDNA. In the 230 s structure, conformational changes directly related to cleavage of the phosphodiester bond between DA4 and DA5 in the DNA are observed.

Bottom Line: The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites.DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion.In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Synchrotron Radiation Research Center, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan.

ABSTRACT
In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII-DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

Show MeSH
Related in: MedlinePlus