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Structural Basis for the Inverted Repeat Preferences of mariner Transposases.

Trubitsyna M, Grey H, Houston DR, Finnegan DJ, Richardson JM - J. Biol. Chem. (2015)

Bottom Line: The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase.A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR.We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end.

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Affiliation: From the Institute of Cell Biology and.

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Comparison of in vitro strand transfer of the left and right Mos1 and Mboumar-9 inverted repeats.A, schematic of the assays. The asterisk indicates the position of the fluorescent label. The size of the products of strand transfer depends on which strand of the target DNA has been attacked. B, denaturing polyacrylamide gel of the Mos1 IR DNA strand transfer products. Lane 1 contains fluorescent labeled markers; lanes 2–6 contain labeled Mos1 IRR DNA; and lanes 7–11 contain labeled Mos1 IRL DNA. Lanes 2 and 7, reactions without transposase; lanes 3 and 8, reactions with no target DNA. The products in lanes 3 and 8 result from integration of IR DNA into the two TA dinucleotides contained within the Mos1 IR DNA sequence. The bands marked by the asterisk are most likely a result of the subsequent integration of the most prominent 37-nt product into target DNA. C, quantification of the percentage of total Mos1 strand transfer products (68 and 40 nt) after a 1.5-h incubation. D, denaturing polyacrylamide gel of the Mboumar-9 IR DNA strand transfer products; lane contents are as described in B except that Mboumar-9 transposase, IRR, and IRL DNA were used. The Mboumar-9 IR DNA sequences contain one TA nucleotide into which other IR DNA molecules can integrate, generating 47- and 51-nt products (lanes 3 and 8). Integration of the most abundant 47-nt product into target DNA would generate an 87-nt strand (marked by asterisk). E, quantification of the percentage of total Mboumar-9 strand transfer products (72 and 44 nt) after a 1.5-h incubation. Error bars in panels C and E indicate the S.D. between 2 and 3 experiments, respectively.
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Figure 4: Comparison of in vitro strand transfer of the left and right Mos1 and Mboumar-9 inverted repeats.A, schematic of the assays. The asterisk indicates the position of the fluorescent label. The size of the products of strand transfer depends on which strand of the target DNA has been attacked. B, denaturing polyacrylamide gel of the Mos1 IR DNA strand transfer products. Lane 1 contains fluorescent labeled markers; lanes 2–6 contain labeled Mos1 IRR DNA; and lanes 7–11 contain labeled Mos1 IRL DNA. Lanes 2 and 7, reactions without transposase; lanes 3 and 8, reactions with no target DNA. The products in lanes 3 and 8 result from integration of IR DNA into the two TA dinucleotides contained within the Mos1 IR DNA sequence. The bands marked by the asterisk are most likely a result of the subsequent integration of the most prominent 37-nt product into target DNA. C, quantification of the percentage of total Mos1 strand transfer products (68 and 40 nt) after a 1.5-h incubation. D, denaturing polyacrylamide gel of the Mboumar-9 IR DNA strand transfer products; lane contents are as described in B except that Mboumar-9 transposase, IRR, and IRL DNA were used. The Mboumar-9 IR DNA sequences contain one TA nucleotide into which other IR DNA molecules can integrate, generating 47- and 51-nt products (lanes 3 and 8). Integration of the most abundant 47-nt product into target DNA would generate an 87-nt strand (marked by asterisk). E, quantification of the percentage of total Mboumar-9 strand transfer products (72 and 44 nt) after a 1.5-h incubation. Error bars in panels C and E indicate the S.D. between 2 and 3 experiments, respectively.

Mentions: To establish whether there is a preference for either the IRR or IRL sequence in the strand transfer step of the transposition reaction, we performed in vitro strand transfer assays using “pre-cleaved” Mos1 IRR or IRL DNA substrates, incorporating a fluorophore at the 5′ end of the 28-nt transferred strand for detection of 68- and 40-nt strand transfer products, as shown schematically in (Fig. 4A). We also performed the assay with Mboumar-9 IRR and IRL DNA substrates and Mboumar-9 transposase; in this case, strand transfer yields labeled products of 72 and 44 nt (Fig. 4A).


Structural Basis for the Inverted Repeat Preferences of mariner Transposases.

Trubitsyna M, Grey H, Houston DR, Finnegan DJ, Richardson JM - J. Biol. Chem. (2015)

Comparison of in vitro strand transfer of the left and right Mos1 and Mboumar-9 inverted repeats.A, schematic of the assays. The asterisk indicates the position of the fluorescent label. The size of the products of strand transfer depends on which strand of the target DNA has been attacked. B, denaturing polyacrylamide gel of the Mos1 IR DNA strand transfer products. Lane 1 contains fluorescent labeled markers; lanes 2–6 contain labeled Mos1 IRR DNA; and lanes 7–11 contain labeled Mos1 IRL DNA. Lanes 2 and 7, reactions without transposase; lanes 3 and 8, reactions with no target DNA. The products in lanes 3 and 8 result from integration of IR DNA into the two TA dinucleotides contained within the Mos1 IR DNA sequence. The bands marked by the asterisk are most likely a result of the subsequent integration of the most prominent 37-nt product into target DNA. C, quantification of the percentage of total Mos1 strand transfer products (68 and 40 nt) after a 1.5-h incubation. D, denaturing polyacrylamide gel of the Mboumar-9 IR DNA strand transfer products; lane contents are as described in B except that Mboumar-9 transposase, IRR, and IRL DNA were used. The Mboumar-9 IR DNA sequences contain one TA nucleotide into which other IR DNA molecules can integrate, generating 47- and 51-nt products (lanes 3 and 8). Integration of the most abundant 47-nt product into target DNA would generate an 87-nt strand (marked by asterisk). E, quantification of the percentage of total Mboumar-9 strand transfer products (72 and 44 nt) after a 1.5-h incubation. Error bars in panels C and E indicate the S.D. between 2 and 3 experiments, respectively.
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Related In: Results  -  Collection

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Figure 4: Comparison of in vitro strand transfer of the left and right Mos1 and Mboumar-9 inverted repeats.A, schematic of the assays. The asterisk indicates the position of the fluorescent label. The size of the products of strand transfer depends on which strand of the target DNA has been attacked. B, denaturing polyacrylamide gel of the Mos1 IR DNA strand transfer products. Lane 1 contains fluorescent labeled markers; lanes 2–6 contain labeled Mos1 IRR DNA; and lanes 7–11 contain labeled Mos1 IRL DNA. Lanes 2 and 7, reactions without transposase; lanes 3 and 8, reactions with no target DNA. The products in lanes 3 and 8 result from integration of IR DNA into the two TA dinucleotides contained within the Mos1 IR DNA sequence. The bands marked by the asterisk are most likely a result of the subsequent integration of the most prominent 37-nt product into target DNA. C, quantification of the percentage of total Mos1 strand transfer products (68 and 40 nt) after a 1.5-h incubation. D, denaturing polyacrylamide gel of the Mboumar-9 IR DNA strand transfer products; lane contents are as described in B except that Mboumar-9 transposase, IRR, and IRL DNA were used. The Mboumar-9 IR DNA sequences contain one TA nucleotide into which other IR DNA molecules can integrate, generating 47- and 51-nt products (lanes 3 and 8). Integration of the most abundant 47-nt product into target DNA would generate an 87-nt strand (marked by asterisk). E, quantification of the percentage of total Mboumar-9 strand transfer products (72 and 44 nt) after a 1.5-h incubation. Error bars in panels C and E indicate the S.D. between 2 and 3 experiments, respectively.
Mentions: To establish whether there is a preference for either the IRR or IRL sequence in the strand transfer step of the transposition reaction, we performed in vitro strand transfer assays using “pre-cleaved” Mos1 IRR or IRL DNA substrates, incorporating a fluorophore at the 5′ end of the 28-nt transferred strand for detection of 68- and 40-nt strand transfer products, as shown schematically in (Fig. 4A). We also performed the assay with Mboumar-9 IRR and IRL DNA substrates and Mboumar-9 transposase; in this case, strand transfer yields labeled products of 72 and 44 nt (Fig. 4A).

Bottom Line: The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase.A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR.We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end.

View Article: PubMed Central - PubMed

Affiliation: From the Institute of Cell Biology and.

Show MeSH
Related in: MedlinePlus