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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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Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and (A) 0.1 U DNase I, (B) 0.15 U S1 nuclease, (C) 1 U exonuclease III (inset shows 10 U), (D) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.
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Figure 2: Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and (A) 0.1 U DNase I, (B) 0.15 U S1 nuclease, (C) 1 U exonuclease III (inset shows 10 U), (D) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.

Mentions: In the case of DNase I, the amounts of residual dsDNA after 10 min in the presence of 20% (w/v) PEG 200, 4000, 8000 and 20000 was estimated to be 12, 4, 10 and 10%, respectively. Thus, PEG 4000 was the most effective of the tested PEGs at enhancing the hydrolysis reaction (Supplementary Figure S3A). For this reason, we further investigated the amount of residual 29-mer dsDNA in the presence of 0–20% (w/v) PEG 4000 versus the time using DNase I at 25°C (Figure 2A). In the absence of PEG 4000, the amount of residual dsDNA after 10 min was estimated to be 87%. In contrast, the amount of residual dsDNA after 10 min in the presence of 5, 10, 15 and 20% (w/v) PEG 4000 was estimated to be 37, 12, 4 and 4%, respectively. These results show that hydrolysis of dsDNA by DNase I is greatly enhanced by PEG.Figure 2.


Regulation of DNA nucleases by molecular crowding.

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

Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and (A) 0.1 U DNase I, (B) 0.15 U S1 nuclease, (C) 1 U exonuclease III (inset shows 10 U), (D) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and (A) 0.1 U DNase I, (B) 0.15 U S1 nuclease, (C) 1 U exonuclease III (inset shows 10 U), (D) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.
Mentions: In the case of DNase I, the amounts of residual dsDNA after 10 min in the presence of 20% (w/v) PEG 200, 4000, 8000 and 20000 was estimated to be 12, 4, 10 and 10%, respectively. Thus, PEG 4000 was the most effective of the tested PEGs at enhancing the hydrolysis reaction (Supplementary Figure S3A). For this reason, we further investigated the amount of residual 29-mer dsDNA in the presence of 0–20% (w/v) PEG 4000 versus the time using DNase I at 25°C (Figure 2A). In the absence of PEG 4000, the amount of residual dsDNA after 10 min was estimated to be 87%. In contrast, the amount of residual dsDNA after 10 min in the presence of 5, 10, 15 and 20% (w/v) PEG 4000 was estimated to be 37, 12, 4 and 4%, respectively. These results show that hydrolysis of dsDNA by DNase I is greatly enhanced by PEG.Figure 2.

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