| 1. |
- Rasila, Tiina S., et al.
(author)
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Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering
- 2018
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 46:9, s. 4649-4661
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Journal article (peer-reviewed)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 trans-posases.
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| 2. |
- Skurnik, Mikael, et al.
(author)
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Characterization of the genome, proteome, and structure of yersiniophage φR1-37
- 2012
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In: Journal of Virology. - 0022-538X. ; 86:23, s. 12625-12642
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Journal article (peer-reviewed)abstract
- The bacteriophage vB_YecM-φR1-37 (φR1-37) is a lytic yersiniophage that can propagate naturally in different Yersinia species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage φR1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of φR1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the φR1-37 capsid consists of hexameric capsomers arranged on a T=27 lattice similar to the bacteriophage φKZ. The tail of φR1-37 has a contractile sheath. We conclude that phage φR1-37 is a representative of a novel phage type that carries the dU-containing genome in a φKZ-like head.
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