title: Target site recognition by a diversity-generating retroelement.
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.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Huatao
authors.initials: H
authors.lastname: Guo
authors.forename: Longping V
authors.initials: LV
authors.lastname: Tse
authors.forename: Angela W
authors.initials: AW
authors.lastname: Nieh
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Czornyj
authors.forename: Steven
authors.initials: S
authors.lastname: Williams
authors.forename: Sabrina
authors.initials: S
authors.lastname: Oukil
authors.forename: Vincent B
authors.initials: VB
authors.lastname: Liu
authors.forename: Jeff F
authors.initials: JF
authors.lastname: Miller
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D001884:Bordetella / Q000235:genetics* / Q000821:virology; D018076:DNA, Complementary / Q000235:genetics; D045566:DNA, Cruciform / Q000235:genetics*; D014644:Genetic Variation; D055029:Inverted Repeat Sequences / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D011506:Proteins / Q000235:genetics; D018626:Retroelements / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: D018076:DNA, Complementary; D045566:DNA, Cruciform; D011506:Proteins; D018626:Retroelements