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Flexibility in MuA Transposase Family Protein Structures: Functional Mapping with Scanning Mutagenesis and Sequence Alignment of Protein Homologues

Rasila, Tiina S.; Vihinen, Mauno LU ; Paulin, Lars; Haapa-Paananen, Saija and Savilahti, Harri (2012) In PLoS ONE 7(5).
Abstract
MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA... (More)
MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains I gamma, II alpha, II beta, and III alpha completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains I alpha and III beta were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general. (Less)
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published
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in
PLoS ONE
volume
7
issue
5
publisher
Public Library of Science
external identifiers
  • wos:000305349600042
  • scopus:84861559424
ISSN
1932-6203
DOI
10.1371/journal.pone.0037922
language
English
LU publication?
yes
id
3e74225c-c263-4e08-99fe-55cc62a33c63 (old id 2890683)
date added to LUP
2012-08-01 08:56:15
date last changed
2017-08-27 04:30:22
@article{3e74225c-c263-4e08-99fe-55cc62a33c63,
  abstract     = {MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains I gamma, II alpha, II beta, and III alpha completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains I alpha and III beta were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.},
  articleno    = {e37922},
  author       = {Rasila, Tiina S. and Vihinen, Mauno and Paulin, Lars and Haapa-Paananen, Saija and Savilahti, Harri},
  issn         = {1932-6203},
  language     = {eng},
  number       = {5},
  publisher    = {Public Library of Science},
  series       = {PLoS ONE},
  title        = {Flexibility in MuA Transposase Family Protein Structures: Functional Mapping with Scanning Mutagenesis and Sequence Alignment of Protein Homologues},
  url          = {http://dx.doi.org/10.1371/journal.pone.0037922},
  volume       = {7},
  year         = {2012},
}