On Diversity Generating Retroelements.

Diversity Generating Retroelements (DGRs) are genetic elements that utilise reverse transcription and site-directed, adenine specific mutagenesis to diversify DNA sequences and the proteins they encode. DGRs have been found encoded on bacterial chromosomes as well as on plasmids and bacteriophage. Most of the research on these elements has concerned the first DGR identified: an element responsible for tropism switching in a temperate bacteriophage of Bordetella.

Bordetella species cause respiratory infections in mammals. The infectious cycle consists of two phases: during Bvg+ phase the bacteria are adapted for colonisation of the respiratory tract whilst in Bvg- they are adapted to ex vivo survival and growth. The phenotypic transition between the phases is associated with major changes in surface structures and secreted proteins. The bacteriophage BPP-1 was identified as having a tropism for the Bvg+ phase, during which it utilised the surface protein Pertactin as it’s receptor. However, BPP-1 was observed to be capable of producing variants that preferentially infected Bvg- phase cells or that were indiscriminate in their phase preference. Comparison of the genomes of the phage variants led to the identification of a region of variability at the 3′ end of a gene mtd(major tropism determinant) and to the characterisation of the BPP-1 DGR.

The BPP-1 DGR

The variable region (VR) is a 134bp sequence, within which nucleotide substitutions occur at 23 discrete positions. The sites of variability are predominantly located in the first two bases of codons, maximising the potential generation of amino acid substitutions. Downstream of VR is a second copy of the 134bp repeat, which is however invariant: the template repeat (TR). Variable sites in VR correspond to adenine residues in TR. When silent substitutions are engineered into TR they are transmitted into VR during tropism switching. When new adenine residues are inserted into TR these too become sites of variability. The TR therefore serves as a donor cassette and VR as the recipient of variable sequence information.

This process of information transfer and selective mutation (‘mutagenic homing’) is mediated by a reverse transcriptase encoded nearby (brt). Another protein of unknown function encoded in the DGR, accessory tropism determinant (atd) is also required. Mutations at brt, atd or TR produce infective phage that are incapable of tropism switching.

Mutagenic homing has been shown to be dependent on short sequences immediately downstream of VR and TR: IMH and IMH* (initiation of mutagenic homing) respectively. These consist of 14bp runs of guanosine and cytosine residues(G/C14 ), followed by a 21bp sequence that differs at five positions between IMH and IMH*. Substitution of IMH* with IMH converts TR into a recipient of diversified sequence information, whereas the opposite substitution eliminates tropism switching. Mutagenic homing has been proposed to occur by a process of target DNA-primed reverse transcription (TPRT). The reverse transcription reaction is primed from either a nick or a double strand break occurring in the G/C14 element of IMH, and is dependent on the rest of IMH as well as potential hairpin/cruciform structure forming short repeat sequences nearby. The 5′ end integration can be mediated by short stretches of homology and is thought to occur by a process of template switching or strand displacement. Mutagenesis has been shown not to occur in the RNA intermediate and so probably occurs during reverse transcription itself. Adenine mutagenesis is thought to be an inherent property of the Brt protein and the related reverse transcriptases encoded in other DGRs.

Homologous DGRs have been identified in nearly 30 diverse bacterial or phage genomes. All of these elements encode related reverse transcriptases and include sequences analogous to TR and VR. Adenine specific mutagenesis is a constant feature of DGR function. The VR encoded variable residues of BPP-1 Mtd are presented on the ligand binding surface using a C-type lectin fold. The structure of this fold is essentially invariant, whilst the ligand binding affinity can differ dramatically. Interestingly, all the VR sequences of DGRs identified thus far are present in C-lec fold encoding sequences. DGRs likely function in a similar manner, however there is diversity in their organisation. TR sequences can be located upstream, downstream or within RTase coding sequences. There is also a dichotomy relating to the presence of atd homologs or genes encoding HRDC (Helicase and RNAseD C terminal) domains. Most interestingly, some DGRs probably diversify multiple VR containing loci.

Most retroelements such as retrotransposons or group II introns are generally considered as ‘selfish DNA’ parasitizing host genomes. DGRs are the first retroelements to obviously confer selective advantage to their hosts. Although their ability to introduce adaptive targeted mutations is within the narrow bounds of diversifying specific C-lec folds they do represent another exception to the ‘central dogma’ of molecular biology and a challenge to the neo-darwinian concept of random mutation.

DGRs could also have important applications in synthetic biology. By engineering a cognate TR and inserting an IMH and related sequences, Jeff Miller’s group (responsible for all this research) were able to diversify antibiotic genes in such a way as to restore function, ie in vivo directed protein evolution.

Watch an excellent 36 min lecture on DGRs by Jeff Miller here

Medhekar, B., & Miller, J. (2007). Diversity-generating retroelements Current Opinion in Microbiology, 10 (4), 388-395 DOI: 10.1016/j.mib.2007.06.004

Liu M, Deora R, Doulatov SR, Gingery M, Eiserling FA, Preston A, Maskell DJ, Simons RW, Cotter PA, Parkhill J, & Miller JF (2002). Reverse transcriptase-mediated tropism switching in Bordetella bacteriophage. Science (New York, N.Y.), 295 (5562), 2091-4 PMID: 11896279

Doulatov S, Hodes A, Dai L, Mandhana N, Liu M, Deora R, Simons RW, Zimmerly S, & Miller JF (2004). Tropism switching in Bordetella bacteriophage defines a family of diversity-generating retroelements. Nature, 431 (7007), 476-81 PMID: 15386016

Guo H, Tse LV, Barbalat R, Sivaamnuaiphorn S, Xu M, Doulatov S, & Miller JF (2008). Diversity-generating retroelement homing regenerates target sequences for repeated rounds of codon rewriting and protein diversification. Molecular cell, 31 (6), 813-23 PMID: 18922465

Guo H, Tse LV, Nieh AW, Czornyj E, Williams S, Oukil S, Liu VB, & Miller JF (2011). Target site recognition by a diversity-generating retroelement. PLoS genetics, 7 (12) PMID: 22194701

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