Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Hydrolysis of plasmid [pcDNA 3.1(+)] DNA by DNase I (0.01 U) in the absence or presence of 20% (w/v) PEG 20000 at 25°C. Lane 1 shows the DNA size marker. Lanes 2, 3, 4, 5, 6 and 7 show 0, 1, 2, 3, 4 and 5 min, respectively, of hydrolysis by DNase I in the absence of PEG 20000. Lanes 8, 9, 10, 11, 12 and 13 show 0, 1, 2, 3, 4 and 5 min, respectively, of hydrolysis by DNase I in the presence of 20% (w/v) PEG 20000. (B) Hydrolysis of a short oligonucleotide DNA (29-mer dsDNA) by DNase I (0.1 U) in the absence or presence of 20% (w/v) PEG 20000 at 25°C. Lanes 1, 2, 3 and 4 show 0, 1, 4 and 10 min, respectively, of hydrolysis by DNase I in the absence of PEG 20000. Lanes 5, 6, 7 and 8 show 0, 1, 4 and 10 min, respectively, of hydrolysis by DNase I in the presence of 20% (w/v) PEG 20000.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1919490&req=5

Figure 1: (A) Hydrolysis of plasmid [pcDNA 3.1(+)] DNA by DNase I (0.01 U) in the absence or presence of 20% (w/v) PEG 20000 at 25°C. Lane 1 shows the DNA size marker. Lanes 2, 3, 4, 5, 6 and 7 show 0, 1, 2, 3, 4 and 5 min, respectively, of hydrolysis by DNase I in the absence of PEG 20000. Lanes 8, 9, 10, 11, 12 and 13 show 0, 1, 2, 3, 4 and 5 min, respectively, of hydrolysis by DNase I in the presence of 20% (w/v) PEG 20000. (B) Hydrolysis of a short oligonucleotide DNA (29-mer dsDNA) by DNase I (0.1 U) in the absence or presence of 20% (w/v) PEG 20000 at 25°C. Lanes 1, 2, 3 and 4 show 0, 1, 4 and 10 min, respectively, of hydrolysis by DNase I in the absence of PEG 20000. Lanes 5, 6, 7 and 8 show 0, 1, 4 and 10 min, respectively, of hydrolysis by DNase I in the presence of 20% (w/v) PEG 20000.

Mentions: We first examined the effect of 20% (w/v) PEG on the hydrolysis of supercoiled plasmid DNA [pcDNA 3.1(+)] by DNase I at 25°C. Figure 1A shows the DNA fragments after electrophoresis on a 0.8% (w/v) agarose gel. The two bands in lanes 2 and 3 correspond to supercoiled and open circular plasmid DNA (32). During the reaction, two distinct bands were observed in the absence of PEG 20000 (lanes 3–7). According to previous observations (32), the slower and faster migrating bands correspond to open circular and linear plasmid DNA, respectively. Although open circular and linear plasmid DNAs were observed, diffuse bands corresponding to products degraded by DNase I were not observed. These results show that, in the absence of PEG, 0.01 U of DNase I was not sufficient for hydrolyzing the plasmid DNA within 5 min. In contrast, in the presence of PEG 20000, the plasmid DNA was cleaved rapidly after 1 min (lane 9), indicating that it was already degraded. These results demonstrate for the first time that molecular crowding significantly enhances the hydrolysis of supercoiled plasmid DNA by DNase I.Figure 1.


Regulation of DNA nucleases by molecular crowding.

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

(A) Hydrolysis of plasmid [pcDNA 3.1(+)] DNA by DNase I (0.01 U) in the absence or presence of 20% (w/v) PEG 20000 at 25°C. Lane 1 shows the DNA size marker. Lanes 2, 3, 4, 5, 6 and 7 show 0, 1, 2, 3, 4 and 5 min, respectively, of hydrolysis by DNase I in the absence of PEG 20000. Lanes 8, 9, 10, 11, 12 and 13 show 0, 1, 2, 3, 4 and 5 min, respectively, of hydrolysis by DNase I in the presence of 20% (w/v) PEG 20000. (B) Hydrolysis of a short oligonucleotide DNA (29-mer dsDNA) by DNase I (0.1 U) in the absence or presence of 20% (w/v) PEG 20000 at 25°C. Lanes 1, 2, 3 and 4 show 0, 1, 4 and 10 min, respectively, of hydrolysis by DNase I in the absence of PEG 20000. Lanes 5, 6, 7 and 8 show 0, 1, 4 and 10 min, respectively, of hydrolysis by DNase I in the presence of 20% (w/v) PEG 20000.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: (A) Hydrolysis of plasmid [pcDNA 3.1(+)] DNA by DNase I (0.01 U) in the absence or presence of 20% (w/v) PEG 20000 at 25°C. Lane 1 shows the DNA size marker. Lanes 2, 3, 4, 5, 6 and 7 show 0, 1, 2, 3, 4 and 5 min, respectively, of hydrolysis by DNase I in the absence of PEG 20000. Lanes 8, 9, 10, 11, 12 and 13 show 0, 1, 2, 3, 4 and 5 min, respectively, of hydrolysis by DNase I in the presence of 20% (w/v) PEG 20000. (B) Hydrolysis of a short oligonucleotide DNA (29-mer dsDNA) by DNase I (0.1 U) in the absence or presence of 20% (w/v) PEG 20000 at 25°C. Lanes 1, 2, 3 and 4 show 0, 1, 4 and 10 min, respectively, of hydrolysis by DNase I in the absence of PEG 20000. Lanes 5, 6, 7 and 8 show 0, 1, 4 and 10 min, respectively, of hydrolysis by DNase I in the presence of 20% (w/v) PEG 20000.
Mentions: We first examined the effect of 20% (w/v) PEG on the hydrolysis of supercoiled plasmid DNA [pcDNA 3.1(+)] by DNase I at 25°C. Figure 1A shows the DNA fragments after electrophoresis on a 0.8% (w/v) agarose gel. The two bands in lanes 2 and 3 correspond to supercoiled and open circular plasmid DNA (32). During the reaction, two distinct bands were observed in the absence of PEG 20000 (lanes 3–7). According to previous observations (32), the slower and faster migrating bands correspond to open circular and linear plasmid DNA, respectively. Although open circular and linear plasmid DNAs were observed, diffuse bands corresponding to products degraded by DNase I were not observed. These results show that, in the absence of PEG, 0.01 U of DNase I was not sufficient for hydrolyzing the plasmid DNA within 5 min. In contrast, in the presence of PEG 20000, the plasmid DNA was cleaved rapidly after 1 min (lane 9), indicating that it was already degraded. These results demonstrate for the first time that molecular crowding significantly enhances the hydrolysis of supercoiled plasmid DNA by DNase I.Figure 1.

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