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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.

View Article: PubMed Central - PubMed

Affiliation: From the Institute of Cell Biology and.

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Inverted repeat preferences of Mos1 and Mboumar-9 transposases for in vitro DNA cleavage of transposon-containing plasmids.A, schematic of the in vitro plasmid DNA cleavage assay and the expected products. The donor DNA plasmid contains a transposon comprising a kanamycin resistance gene (kanR) flanked by inverted repeats (black arrows), a chloramphenicol resistance gene (camR), and the oriR6K origin of replication. B, agarose gel of the products of in vitro IR DNA cleavage. Lane 1, 1-kb DNA ladder of markers (M); lane 2, pEPMosLL linearized with XbaI; lane 3, pEPMboLL digested with SacI to excise the transposon; lane 4, supercoiled (sc) plasmid. Lanes 5–7, cleavage of the Mos1 transposon containing two left IRs (LL), one left and one right IR (LR), or two right IRs (RR). Lanes 8–10, cleavage of Mboumar-9 transposons. C, quantification of the percentage of plasmid backbone released (as a proportion of the total intensity of the lane) in each of the reactions in lanes 5–10 above. The error bars indicate the S.D. between 4 independent measurements.
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Figure 3: Inverted repeat preferences of Mos1 and Mboumar-9 transposases for in vitro DNA cleavage of transposon-containing plasmids.A, schematic of the in vitro plasmid DNA cleavage assay and the expected products. The donor DNA plasmid contains a transposon comprising a kanamycin resistance gene (kanR) flanked by inverted repeats (black arrows), a chloramphenicol resistance gene (camR), and the oriR6K origin of replication. B, agarose gel of the products of in vitro IR DNA cleavage. Lane 1, 1-kb DNA ladder of markers (M); lane 2, pEPMosLL linearized with XbaI; lane 3, pEPMboLL digested with SacI to excise the transposon; lane 4, supercoiled (sc) plasmid. Lanes 5–7, cleavage of the Mos1 transposon containing two left IRs (LL), one left and one right IR (LR), or two right IRs (RR). Lanes 8–10, cleavage of Mboumar-9 transposons. C, quantification of the percentage of plasmid backbone released (as a proportion of the total intensity of the lane) in each of the reactions in lanes 5–10 above. The error bars indicate the S.D. between 4 independent measurements.

Mentions: To establish whether there is an optimal arrangement of IRs for transposon excision, we performed in vitro cleavage reactions (20) (Fig. 3A). Transposons containing either the natural combination of IRR and IRL or two copies of IRR or IRL were created for both Mos1 and Mboumar-9. For Mos1, we found the highest level of transposon excision (9.1%) from the donor plasmid backbone using a transposon containing two Mos1 IRRs (Fig. 3, B and C). Mboumar-9 also had a preference toward one of the inverted repeats, in this case IRL with 10.7% excision (Fig. 3, B and C). In the most active excision reactions (Mos1 with IRR and Mboumar-9 with IRL), excised transposon bands of 1.3 kb can be observed.


Structural Basis for the Inverted Repeat Preferences of mariner Transposases.

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

Inverted repeat preferences of Mos1 and Mboumar-9 transposases for in vitro DNA cleavage of transposon-containing plasmids.A, schematic of the in vitro plasmid DNA cleavage assay and the expected products. The donor DNA plasmid contains a transposon comprising a kanamycin resistance gene (kanR) flanked by inverted repeats (black arrows), a chloramphenicol resistance gene (camR), and the oriR6K origin of replication. B, agarose gel of the products of in vitro IR DNA cleavage. Lane 1, 1-kb DNA ladder of markers (M); lane 2, pEPMosLL linearized with XbaI; lane 3, pEPMboLL digested with SacI to excise the transposon; lane 4, supercoiled (sc) plasmid. Lanes 5–7, cleavage of the Mos1 transposon containing two left IRs (LL), one left and one right IR (LR), or two right IRs (RR). Lanes 8–10, cleavage of Mboumar-9 transposons. C, quantification of the percentage of plasmid backbone released (as a proportion of the total intensity of the lane) in each of the reactions in lanes 5–10 above. The error bars indicate the S.D. between 4 independent measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4505599&req=5

Figure 3: Inverted repeat preferences of Mos1 and Mboumar-9 transposases for in vitro DNA cleavage of transposon-containing plasmids.A, schematic of the in vitro plasmid DNA cleavage assay and the expected products. The donor DNA plasmid contains a transposon comprising a kanamycin resistance gene (kanR) flanked by inverted repeats (black arrows), a chloramphenicol resistance gene (camR), and the oriR6K origin of replication. B, agarose gel of the products of in vitro IR DNA cleavage. Lane 1, 1-kb DNA ladder of markers (M); lane 2, pEPMosLL linearized with XbaI; lane 3, pEPMboLL digested with SacI to excise the transposon; lane 4, supercoiled (sc) plasmid. Lanes 5–7, cleavage of the Mos1 transposon containing two left IRs (LL), one left and one right IR (LR), or two right IRs (RR). Lanes 8–10, cleavage of Mboumar-9 transposons. C, quantification of the percentage of plasmid backbone released (as a proportion of the total intensity of the lane) in each of the reactions in lanes 5–10 above. The error bars indicate the S.D. between 4 independent measurements.
Mentions: To establish whether there is an optimal arrangement of IRs for transposon excision, we performed in vitro cleavage reactions (20) (Fig. 3A). Transposons containing either the natural combination of IRR and IRL or two copies of IRR or IRL were created for both Mos1 and Mboumar-9. For Mos1, we found the highest level of transposon excision (9.1%) from the donor plasmid backbone using a transposon containing two Mos1 IRRs (Fig. 3, B and C). Mboumar-9 also had a preference toward one of the inverted repeats, in this case IRL with 10.7% excision (Fig. 3, B and C). In the most active excision reactions (Mos1 with IRR and Mboumar-9 with IRL), excised transposon bands of 1.3 kb can be observed.

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