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The folding of the specific DNA recognition subdomain of the sleeping beauty transposase is temperature-dependent and is required for its binding to the transposon DNA.

Leighton GO, Konnova TA, Idiyatullin B, Hurr SH, Zuev YF, Nesmelova IV - PLoS ONE (2014)

Bottom Line: Here, we show that only the folded conformation of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain, binds to the transposon DNA.Furthermore, we show that the PAI subdomain is well folded at low temperatures, but the presence of unfolded conformation gradually increases at temperatures above 15°C, suggesting that the choice of temperature may be important for the optimal transposase activity.Overall, the results provide a molecular-level insight into the DNA recognition by the Sleeping Beauty transposase.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Optical Science, University of North Carolina, Charlotte, North Carolina, United States of America.

ABSTRACT
The reaction of DNA transposition begins when the transposase enzyme binds to the transposon DNA. Sleeping Beauty is a member of the mariner family of DNA transposons. Although it is an important tool in genetic applications and has been adapted for human gene therapy, its molecular mechanism remains obscure. Here, we show that only the folded conformation of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain, binds to the transposon DNA. Furthermore, we show that the PAI subdomain is well folded at low temperatures, but the presence of unfolded conformation gradually increases at temperatures above 15°C, suggesting that the choice of temperature may be important for the optimal transposase activity. Overall, the results provide a molecular-level insight into the DNA recognition by the Sleeping Beauty transposase.

No MeSH data available.


Related in: MedlinePlus

Self-diffusion coefficients.The PAI self-diffusion coefficient at pH 5.0 (squares) and 7.0 (circles) are plotted vs. temperature over the range from 5 to 35°C. Protein samples were prepared in 25 mM sodium phosphate buffer using 100% D2O. The temperature dependence of the self-diffusion coefficient of BPTI (stars) is shown for comparison. Solid lines represent fits of Arrhenius dependence of the self-diffusion coefficient to experimental data.
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pone-0112114-g003: Self-diffusion coefficients.The PAI self-diffusion coefficient at pH 5.0 (squares) and 7.0 (circles) are plotted vs. temperature over the range from 5 to 35°C. Protein samples were prepared in 25 mM sodium phosphate buffer using 100% D2O. The temperature dependence of the self-diffusion coefficient of BPTI (stars) is shown for comparison. Solid lines represent fits of Arrhenius dependence of the self-diffusion coefficient to experimental data.

Mentions: According to the Stokes-Einstein equation D =  kBT/6πηR, the self-diffusion coefficient is inversely proportional to the radius, R, of the diffusing species in solution; hence, it was used to determine whether the aggregation state of the PAI subdomain changes with temperature. Other quantities in the Stokes-Einstein equation include the Boltzmann constant kB and the viscosity of pure solvent η (e.g., D2O). In the absence of processes that could lead to the change of protein size with temperature, i.e. protein aggregation or unfolding, the temperature dependence of D is determined only by the temperature dependence of the viscosity η of D2O. Accordingly, it is expected to follow the Arrhenius relation with a slope reflecting the activation energy of the self-diffusion of water (5 kcal/mol) [28]. Figure 3 shows the temperature dependence of the PAI self-diffusion coefficient at pH 5.0 (squares) and 7.0 (circles) over the temperature range 5–35°C. The temperature dependence of the self-diffusion coefficient of bovine pancreatic trypsin inhibitor (BPTI), which has a comparable molecular weight (6.5 kDa for BPTI vs. 6.9 kDa for PAI) and remains monomeric and folded [29] in the temperature range from 10 to 42°C, was also measured and is shown in Figure 3 for comparison (stars). Solid lines represent fits of Arrhenius dependence of the self-diffusion coefficient to experimental data. The fit for BPTI was done in the interval of temperatures corresponding to its monomeric state. Several conclusions are apparent from Figure 3. (1) The self-diffusion coefficients of the PAI subdomain measured at pH 5.0 and pH 7.0 are different at all temperatures. (2) The temperature dependence of PAI self-diffusion coefficient is linear throughout the entire temperature range, with the slope corresponding to the activation energy of 5.7 kcal/mol at pH 7.0 and 5.8 kcal/mol at pH 5.0. (3) The self-diffusion coefficient of the PAI subdomain is close to the self-diffusion coefficient of BPTI by magnitude, with the self-diffusion coefficient somewhat lower and higher than that of BPTI at pH 5.0 and 7.0, respectively. (4) The slopes of the temperature dependence of PAI self-diffusion coefficient are slightly steeper than the slope of the BPTI self-diffusion coefficient (5.4 kcal/mol) at both pH values.


The folding of the specific DNA recognition subdomain of the sleeping beauty transposase is temperature-dependent and is required for its binding to the transposon DNA.

Leighton GO, Konnova TA, Idiyatullin B, Hurr SH, Zuev YF, Nesmelova IV - PLoS ONE (2014)

Self-diffusion coefficients.The PAI self-diffusion coefficient at pH 5.0 (squares) and 7.0 (circles) are plotted vs. temperature over the range from 5 to 35°C. Protein samples were prepared in 25 mM sodium phosphate buffer using 100% D2O. The temperature dependence of the self-diffusion coefficient of BPTI (stars) is shown for comparison. Solid lines represent fits of Arrhenius dependence of the self-diffusion coefficient to experimental data.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222973&req=5

pone-0112114-g003: Self-diffusion coefficients.The PAI self-diffusion coefficient at pH 5.0 (squares) and 7.0 (circles) are plotted vs. temperature over the range from 5 to 35°C. Protein samples were prepared in 25 mM sodium phosphate buffer using 100% D2O. The temperature dependence of the self-diffusion coefficient of BPTI (stars) is shown for comparison. Solid lines represent fits of Arrhenius dependence of the self-diffusion coefficient to experimental data.
Mentions: According to the Stokes-Einstein equation D =  kBT/6πηR, the self-diffusion coefficient is inversely proportional to the radius, R, of the diffusing species in solution; hence, it was used to determine whether the aggregation state of the PAI subdomain changes with temperature. Other quantities in the Stokes-Einstein equation include the Boltzmann constant kB and the viscosity of pure solvent η (e.g., D2O). In the absence of processes that could lead to the change of protein size with temperature, i.e. protein aggregation or unfolding, the temperature dependence of D is determined only by the temperature dependence of the viscosity η of D2O. Accordingly, it is expected to follow the Arrhenius relation with a slope reflecting the activation energy of the self-diffusion of water (5 kcal/mol) [28]. Figure 3 shows the temperature dependence of the PAI self-diffusion coefficient at pH 5.0 (squares) and 7.0 (circles) over the temperature range 5–35°C. The temperature dependence of the self-diffusion coefficient of bovine pancreatic trypsin inhibitor (BPTI), which has a comparable molecular weight (6.5 kDa for BPTI vs. 6.9 kDa for PAI) and remains monomeric and folded [29] in the temperature range from 10 to 42°C, was also measured and is shown in Figure 3 for comparison (stars). Solid lines represent fits of Arrhenius dependence of the self-diffusion coefficient to experimental data. The fit for BPTI was done in the interval of temperatures corresponding to its monomeric state. Several conclusions are apparent from Figure 3. (1) The self-diffusion coefficients of the PAI subdomain measured at pH 5.0 and pH 7.0 are different at all temperatures. (2) The temperature dependence of PAI self-diffusion coefficient is linear throughout the entire temperature range, with the slope corresponding to the activation energy of 5.7 kcal/mol at pH 7.0 and 5.8 kcal/mol at pH 5.0. (3) The self-diffusion coefficient of the PAI subdomain is close to the self-diffusion coefficient of BPTI by magnitude, with the self-diffusion coefficient somewhat lower and higher than that of BPTI at pH 5.0 and 7.0, respectively. (4) The slopes of the temperature dependence of PAI self-diffusion coefficient are slightly steeper than the slope of the BPTI self-diffusion coefficient (5.4 kcal/mol) at both pH values.

Bottom Line: Here, we show that only the folded conformation of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain, binds to the transposon DNA.Furthermore, we show that the PAI subdomain is well folded at low temperatures, but the presence of unfolded conformation gradually increases at temperatures above 15°C, suggesting that the choice of temperature may be important for the optimal transposase activity.Overall, the results provide a molecular-level insight into the DNA recognition by the Sleeping Beauty transposase.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Optical Science, University of North Carolina, Charlotte, North Carolina, United States of America.

ABSTRACT
The reaction of DNA transposition begins when the transposase enzyme binds to the transposon DNA. Sleeping Beauty is a member of the mariner family of DNA transposons. Although it is an important tool in genetic applications and has been adapted for human gene therapy, its molecular mechanism remains obscure. Here, we show that only the folded conformation of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain, binds to the transposon DNA. Furthermore, we show that the PAI subdomain is well folded at low temperatures, but the presence of unfolded conformation gradually increases at temperatures above 15°C, suggesting that the choice of temperature may be important for the optimal transposase activity. Overall, the results provide a molecular-level insight into the DNA recognition by the Sleeping Beauty transposase.

No MeSH data available.


Related in: MedlinePlus