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Regulation of DNA nucleases by molecular crowding.

Sasaki Y, Miyoshi D, Sugimoto N - Nucleic Acids Res. (2007)

Bottom Line: We found that the hydrolysis of a 29-mer double-stranded DNA by the endonucleases DNase I and S1 nuclease was substantially enhanced by molecular crowding using polyethylene glycol (PEG); however, molecular crowding had little effect on hydrolysis by exo III and exo I exonucleases.In contrast, molecular crowding did not significantly affect the Michaelis constant of DNase I or exonuclease I.These results indicate that molecular crowding has different effects on the catalytic activities of exonucleases and endonucleases.

View Article: PubMed Central - PubMed

Affiliation: Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Fine Co., Ltd., 5-7-8 Shimoshinjo, Higashiyodogawa-ku, Osaka.

ABSTRACT
Here, we examined the effects of molecular crowding on the function, structure and stability of nucleases. We found that the hydrolysis of a 29-mer double-stranded DNA by the endonucleases DNase I and S1 nuclease was substantially enhanced by molecular crowding using polyethylene glycol (PEG); however, molecular crowding had little effect on hydrolysis by exo III and exo I exonucleases. Moreover, kinetic analysis showed that the maximum velocity for the reaction of DNase I at 25 degrees C was increased from 0.1 to 2.7 microM/min by molecular crowding with 20% (w/v) PEG, whereas that of exonuclease I at 37 degrees C decreased from 2.2 to 0.4 microM/min. In contrast, molecular crowding did not significantly affect the Michaelis constant of DNase I or exonuclease I. These results indicate that molecular crowding has different effects on the catalytic activities of exonucleases and endonucleases.

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(A) CD spectra for 0.02 mg/ml DNase I in the absence (dotted line) and presence (solid line) of 20% (w/v) PEG 4000 at 25°C. (B) Thermal denaturation curves for DNase I in the absence (dotted line) and presence (solid line) of 20% (w/v) PEG 4000. The changes in the ellipticity were monitored at 222 nm. All measurements were performed in 50 mM HEPES (pH 7.2).
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Figure 3: (A) CD spectra for 0.02 mg/ml DNase I in the absence (dotted line) and presence (solid line) of 20% (w/v) PEG 4000 at 25°C. (B) Thermal denaturation curves for DNase I in the absence (dotted line) and presence (solid line) of 20% (w/v) PEG 4000. The changes in the ellipticity were monitored at 222 nm. All measurements were performed in 50 mM HEPES (pH 7.2).

Mentions: Although we found that molecular crowding enhances endonuclease activity rather than exonuclease activity, the origin of the difference in the effects remains unclear. Because we already demonstrated that the structure and stability of the substrate DNAs are not critical factors in the molecular crowding effect, we examined whether molecular crowding affects the thermodynamic stability of the nucleases. We first compared the structure and stability of an endonuclease (DNase I) in dilute and molecular crowding conditions by CD. Figure 3A shows CD spectra of DNase I in the absence and presence of 20% (w/v) PEG 4000 at 25°C. Both CD spectra have negative peaks around 215 and 208 nm and a positive peak around 198 nm, showing that α-helices are the dominant structure in DNase I in dilute and molecular crowding conditions. These results are identical with those reported by Ajitai and Venyaminov (38). In addition, PEG 4000 did not cause a peak shift, indicating that molecular crowding did not affect the secondary structure of DNase I. On the other hand, molecular crowding strongly affected the stability of the DNase I structure. Figure 3B shows melting curves for the α-helical DNase I structure in the absence and presence of 20% (w/v) PEG 4000. The melting temperature (Tm) of the DNase I structure in the absence of PEG 4000 was estimated to be 60°C. Surprisingly, the Tm of the DNase I structure was >80°C in the presence of 20% (w/v) PEG 4000. This demonstrates that molecular crowding stabilizes the structure of DNase I.Figure 3.


Regulation of DNA nucleases by molecular crowding.

Sasaki Y, Miyoshi D, Sugimoto N - Nucleic Acids Res. (2007)

(A) CD spectra for 0.02 mg/ml DNase I in the absence (dotted line) and presence (solid line) of 20% (w/v) PEG 4000 at 25°C. (B) Thermal denaturation curves for DNase I in the absence (dotted line) and presence (solid line) of 20% (w/v) PEG 4000. The changes in the ellipticity were monitored at 222 nm. All measurements were performed in 50 mM HEPES (pH 7.2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1919490&req=5

Figure 3: (A) CD spectra for 0.02 mg/ml DNase I in the absence (dotted line) and presence (solid line) of 20% (w/v) PEG 4000 at 25°C. (B) Thermal denaturation curves for DNase I in the absence (dotted line) and presence (solid line) of 20% (w/v) PEG 4000. The changes in the ellipticity were monitored at 222 nm. All measurements were performed in 50 mM HEPES (pH 7.2).
Mentions: Although we found that molecular crowding enhances endonuclease activity rather than exonuclease activity, the origin of the difference in the effects remains unclear. Because we already demonstrated that the structure and stability of the substrate DNAs are not critical factors in the molecular crowding effect, we examined whether molecular crowding affects the thermodynamic stability of the nucleases. We first compared the structure and stability of an endonuclease (DNase I) in dilute and molecular crowding conditions by CD. Figure 3A shows CD spectra of DNase I in the absence and presence of 20% (w/v) PEG 4000 at 25°C. Both CD spectra have negative peaks around 215 and 208 nm and a positive peak around 198 nm, showing that α-helices are the dominant structure in DNase I in dilute and molecular crowding conditions. These results are identical with those reported by Ajitai and Venyaminov (38). In addition, PEG 4000 did not cause a peak shift, indicating that molecular crowding did not affect the secondary structure of DNase I. On the other hand, molecular crowding strongly affected the stability of the DNase I structure. Figure 3B shows melting curves for the α-helical DNase I structure in the absence and presence of 20% (w/v) PEG 4000. The melting temperature (Tm) of the DNase I structure in the absence of PEG 4000 was estimated to be 60°C. Surprisingly, the Tm of the DNase I structure was >80°C in the presence of 20% (w/v) PEG 4000. This demonstrates that molecular crowding stabilizes the structure of DNase I.Figure 3.

Bottom Line: We found that the hydrolysis of a 29-mer double-stranded DNA by the endonucleases DNase I and S1 nuclease was substantially enhanced by molecular crowding using polyethylene glycol (PEG); however, molecular crowding had little effect on hydrolysis by exo III and exo I exonucleases.In contrast, molecular crowding did not significantly affect the Michaelis constant of DNase I or exonuclease I.These results indicate that molecular crowding has different effects on the catalytic activities of exonucleases and endonucleases.

View Article: PubMed Central - PubMed

Affiliation: Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Fine Co., Ltd., 5-7-8 Shimoshinjo, Higashiyodogawa-ku, Osaka.

ABSTRACT
Here, we examined the effects of molecular crowding on the function, structure and stability of nucleases. We found that the hydrolysis of a 29-mer double-stranded DNA by the endonucleases DNase I and S1 nuclease was substantially enhanced by molecular crowding using polyethylene glycol (PEG); however, molecular crowding had little effect on hydrolysis by exo III and exo I exonucleases. Moreover, kinetic analysis showed that the maximum velocity for the reaction of DNase I at 25 degrees C was increased from 0.1 to 2.7 microM/min by molecular crowding with 20% (w/v) PEG, whereas that of exonuclease I at 37 degrees C decreased from 2.2 to 0.4 microM/min. In contrast, molecular crowding did not significantly affect the Michaelis constant of DNase I or exonuclease I. These results indicate that molecular crowding has different effects on the catalytic activities of exonucleases and endonucleases.

Show MeSH
Related in: MedlinePlus