Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering

Tiina S Rasila, Elsi Pulkkinen, Saija Kiljunen, Saija Haapa-Paananen, Maria I Pajunen, Anu Salminen, Lars Paulin, Mauno Vihinen, Phoebe A Rice, Harri Savilahti

Research output: Contribution to journalArticleScientificpeer-review

Abstract

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.
Original languageEnglish
JournalNucleic Acids Research
Volume46
Issue number9
Pages (from-to)4649-4661
Number of pages12
ISSN0305-1048
DOIs
Publication statusPublished - 18 May 2018
MoE publication typeA1 Journal article-refereed

Fields of Science

  • 1182 Biochemistry, cell and molecular biology

Cite this

Rasila, Tiina S ; Pulkkinen, Elsi ; Kiljunen, Saija ; Haapa-Paananen, Saija ; Pajunen, Maria I ; Salminen, Anu ; Paulin, Lars ; Vihinen, Mauno ; Rice, Phoebe A ; Savilahti, Harri. / Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering. In: Nucleic Acids Research. 2018 ; Vol. 46, No. 9. pp. 4649-4661.
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abstract = "The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.",
keywords = "1182 Biochemistry, cell and molecular biology",
author = "Rasila, {Tiina S} and Elsi Pulkkinen and Saija Kiljunen and Saija Haapa-Paananen and Pajunen, {Maria I} and Anu Salminen and Lars Paulin and Mauno Vihinen and Rice, {Phoebe A} and Harri Savilahti",
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Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering. / Rasila, Tiina S; Pulkkinen, Elsi; Kiljunen, Saija; Haapa-Paananen, Saija; Pajunen, Maria I; Salminen, Anu; Paulin, Lars; Vihinen, Mauno; Rice, Phoebe A; Savilahti, Harri.

In: Nucleic Acids Research, Vol. 46, No. 9, 18.05.2018, p. 4649-4661.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering

AU - Rasila, Tiina S

AU - Pulkkinen, Elsi

AU - Kiljunen, Saija

AU - Haapa-Paananen, Saija

AU - Pajunen, Maria I

AU - Salminen, Anu

AU - Paulin, Lars

AU - Vihinen, Mauno

AU - Rice, Phoebe A

AU - Savilahti, Harri

PY - 2018/5/18

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N2 - The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.

AB - The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.

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