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      Target Site Recognition by a Diversity-Generating Retroelement

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          Abstract

          Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.

          Author Summary

          Diversity-generating retroelements function through a unique, reverse transcriptase–mediated “copy and replace” mechanism that enables repeated rounds of protein diversification, selection, and optimization. The ability of DGRs to introduce targeted diversity into protein-coding DNA sequences has the potential to dramatically accelerate the evolution of adaptive traits. The utility of these elements in nature is underscored by their widespread distribution throughout the bacterial domain. Here we define DNA sequences and structures that are necessary and sufficient to direct the diversification machinery to specified target sequences. In addition to providing mechanistic insights into conserved features of DGR activity, our results provide a blueprint for the use of DGRs for a broad range of protein engineering applications.

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          Most cited references24

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          Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition.

          R2 is a non-LTR retrotransposable element that inserts at a specific site in the 28S rRNA genes of most insects. We have expressed the open reading frame of the R2 element from Bombyx mori, R2Bm, in E. coli and shown that it encodes both sequence-specific endonuclease and reverse transcriptase activities. The R2 protein makes a specific nick in one of the DNA strands at the insertion site and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of its RNA transcript. After reverse transcription, cleavage of the second DNA strand occurs. A similar mechanism of insertion may be used by other non-LTR retrotransposable elements as well as short interspersed nucleotide elements.
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            Mobile group II introns.

            Mobile group II introns, found in bacterial and organellar genomes, are both catalytic RNAs and retrotransposable elements. They use an extraordinary mobility mechanism in which the excised intron RNA reverse splices directly into a DNA target site and is then reverse transcribed by the intron-encoded protein. After DNA insertion, the introns remove themselves by protein-assisted, autocatalytic RNA splicing, thereby minimizing host damage. Here we discuss the experimental basis for our current understanding of group II intron mobility mechanisms, beginning with genetic observations in yeast mitochondria, and culminating with a detailed understanding of molecular mechanisms shared by organellar and bacterial group II introns. We also discuss recently discovered links between group II intron mobility and DNA replication, new insights into group II intron evolution arising from bacterial genome sequencing, and the evolutionary relationship between group II introns and both eukaryotic spliceosomal introns and non-LTR-retrotransposons. Finally, we describe the development of mobile group II introns into gene-targeting vectors, "targetrons," which have programmable target specificity.
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              A simple chemically defined medium for the production of phase I Bordetella pertussis.

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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                December 2011
                December 2011
                15 December 2011
                : 7
                : 12
                : e1002414
                Affiliations
                [1 ]Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America
                [2 ]AvidBiotics Corporation, South San Francisco, California, United States of America
                [3 ]The Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America
                Agency for Science, Technology, and Research, Singapore
                Author notes

                Conceived and designed the experiments: HG JFM. Performed the experiments: HG LVT AWN EC SW SO VBL. Analyzed the data: HG JFM. Wrote the paper: HG JFM.

                Article
                PGENETICS-D-11-01762
                10.1371/journal.pgen.1002414
                3240598
                22194701
                373ce614-61bf-4167-9b32-cb2b1c4d8c46
                Guo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 17 August 2011
                : 27 October 2011
                Page count
                Pages: 16
                Categories
                Research Article
                Biology
                Biochemistry
                Nucleic Acids
                DNA
                DNA structure
                Biotechnology
                Applied Microbiology
                Evolutionary Biology
                Evolutionary Processes
                Adaptation
                Mutation
                Organismal Evolution
                Microbial Evolution
                Evolutionary Genetics
                Genomic Evolution
                Genetics
                Genetic Mutation
                Mutagenesis
                Microbiology
                Bacteriology
                Bacterial Evolution
                Applied Microbiology
                Microbial Evolution
                Microbial Mutation
                Molecular Cell Biology
                Nucleic Acids
                DNA
                DNA structure
                Transposons
                Retrotransposons

                Genetics
                Genetics

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