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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

Intrinsic tyrosine fluorescence.(Left panel) The location of Y46 on the cartoon representation of the PAI subdomain (PDB code 2m8e [7]) is shown. (Right panel) The integral fluorescence of Y46 at pH 5.0 (squares) and pH 7.0 (circles) is plotted vs. temperature. Shown data is the average of three independent experiments. Error bars in many cases do not exceed the size of the symbol. Solid lines represent best fits of experimental data.
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pone-0112114-g002: Intrinsic tyrosine fluorescence.(Left panel) The location of Y46 on the cartoon representation of the PAI subdomain (PDB code 2m8e [7]) is shown. (Right panel) The integral fluorescence of Y46 at pH 5.0 (squares) and pH 7.0 (circles) is plotted vs. temperature. Shown data is the average of three independent experiments. Error bars in many cases do not exceed the size of the symbol. Solid lines represent best fits of experimental data.

Mentions: Intrinsic tyrosine fluorescence was used to monitor the change of the PAI subdomain folding state with temperature. Proteins derive their intrinsic fluorescence from the chromophores phenylalanine, tyrosine, and tryptophan. The PAI subdomain contains only one tyrosine (Y46) and does not have any phenylalanine or tryptophan residues. The side chain of Y46 is oriented towards the interior of the protein (Figure 2, left panel), and thus should be sensitive to the PAI subdomain unfolding. The total integral fluorescence intensity as a function of temperature is plotted in Figure 2 (right panel) for pH values of 5.0 and 7.0 using a semi-logarithmic scale. At pH 5.0, the temperature dependence of Y46 fluorescence is linear, as expected, because the PAI subdomain remains unfolded and the environment of Y46 does not change. At pH 7.0, the transition between 18 and 28°C is observed, indicating that there is a change in the Y46 environment due to PAI unfolding, in agreement with our [1H,15N]-HSQC data shown in Figure 1. We note that the effect is not very strong. This is likely due to the fact that even in a folded state Y46 has good water accessibility due to the small size and flexibility of the PAI subdomain.


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)

Intrinsic tyrosine fluorescence.(Left panel) The location of Y46 on the cartoon representation of the PAI subdomain (PDB code 2m8e [7]) is shown. (Right panel) The integral fluorescence of Y46 at pH 5.0 (squares) and pH 7.0 (circles) is plotted vs. temperature. Shown data is the average of three independent experiments. Error bars in many cases do not exceed the size of the symbol. Solid lines represent best fits of experimental data.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112114-g002: Intrinsic tyrosine fluorescence.(Left panel) The location of Y46 on the cartoon representation of the PAI subdomain (PDB code 2m8e [7]) is shown. (Right panel) The integral fluorescence of Y46 at pH 5.0 (squares) and pH 7.0 (circles) is plotted vs. temperature. Shown data is the average of three independent experiments. Error bars in many cases do not exceed the size of the symbol. Solid lines represent best fits of experimental data.
Mentions: Intrinsic tyrosine fluorescence was used to monitor the change of the PAI subdomain folding state with temperature. Proteins derive their intrinsic fluorescence from the chromophores phenylalanine, tyrosine, and tryptophan. The PAI subdomain contains only one tyrosine (Y46) and does not have any phenylalanine or tryptophan residues. The side chain of Y46 is oriented towards the interior of the protein (Figure 2, left panel), and thus should be sensitive to the PAI subdomain unfolding. The total integral fluorescence intensity as a function of temperature is plotted in Figure 2 (right panel) for pH values of 5.0 and 7.0 using a semi-logarithmic scale. At pH 5.0, the temperature dependence of Y46 fluorescence is linear, as expected, because the PAI subdomain remains unfolded and the environment of Y46 does not change. At pH 7.0, the transition between 18 and 28°C is observed, indicating that there is a change in the Y46 environment due to PAI unfolding, in agreement with our [1H,15N]-HSQC data shown in Figure 1. We note that the effect is not very strong. This is likely due to the fact that even in a folded state Y46 has good water accessibility due to the small size and flexibility of the PAI subdomain.

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