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Diversity-generating Retroelements in Phage and Bacterial Genomes

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  • Authors: Huatao Guo1, Diego Arambula2, Partho Ghosh3, Jeff F. Miller4
  • Editors: Alan Lambowitz6, Nancy Craig7
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine Columbia, MO 65212; 2: Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095; 3: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093; 4: Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095; 5: The California NanoSystems Institute, University of California at Los Angeles, Los Angeles, CA 90095; 6: University of Texas, Austin, TX; 7: Johns Hopkins University, Baltimore, MD
  • Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0029-2014
  • Received 30 May 2014 Accepted 17 July 2014 Published 05 December 2014
  • Jeff F. Miller, jfmiller@ucla.edu, Huatao Guo, guohua@missouri.edu
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  • Abstract:

    Diversity-generating retroelements (DGRs) are DNA diversification machines found in diverse bacterial and bacteriophage genomes that accelerate the evolution of ligand–receptor interactions. Diversification results from a unidirectional transfer of sequence information from an invariant template repeat (TR) to a variable repeat (VR) located in a protein-encoding gene. Information transfer is coupled to site-specific mutagenesis in a process called mutagenic homing, which occurs through an RNA intermediate and is catalyzed by a unique, DGR-encoded reverse transcriptase that converts adenine residues in the TR into random nucleotides in the VR. In the prototype DGR found in the bacteriophage BPP-1, the variable protein Mtd is responsible for phage receptor recognition. VR diversification enables progeny phage to switch tropism, accelerating their adaptation to changes in sequence or availability of host cell-surface molecules for infection. Since their discovery, hundreds of DGRs have been identified, and their functions are just beginning to be understood. VR-encoded residues of many DGR-diversified proteins are displayed in the context of a C-type lectin fold, although other scaffolds, including the immunoglobulin fold, may also be used. DGR homing is postulated to occur through a specialized target DNA-primed reverse transcription mechanism that allows repeated rounds of diversification and selection, and the ability to engineer DGRs to target heterologous genes suggests applications for bioengineering. This chapter provides a comprehensive review of our current understanding of this newly discovered family of beneficial retroelements.

  • Citation: Guo H, Arambula D, Ghosh P, Miller J. 2014. Diversity-generating Retroelements in Phage and Bacterial Genomes. Microbiol Spectrum 2(6):MDNA3-0029-2014. doi:10.1128/microbiolspec.MDNA3-0029-2014.

Key Concept Ranking

Mobile Genetic Elements
0.7011057
Human immunodeficiency virus 1
0.47367355
Type III Secretion System
0.41362983
0.7011057

References

1. Vink C, Rudenko G, Seifert HS. 2012. Microbial antigenic variation mediated by homologous DNA recombination. FEMS Microbiol Rev 36:917–948. [PubMed]
2. Agrawal A, Eastman QM, Schatz DG. 1998. Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394:744–751. [PubMed][CrossRef]
3. Kapitonov VV, Jurka J. 2005. RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons. PLoS Biol 3:e181. [PubMed][CrossRef]
4. Hencken CG, Li X, Craig NL. 2012. Functional characterization of an active Rag-like transposase. Nat Struct Mol Biol 19:834–836. [PubMed][CrossRef]
5. 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 295:2091–2094. [PubMed][CrossRef]
6. 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 431:476–481. [PubMed][CrossRef]
7. 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. Mol Cell 31:813–823. [PubMed][CrossRef]
8. Melvin JA, Scheller EV, Miller JF, Cotter PA. 2014. Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol 12:274–288. [PubMed][CrossRef]
9. Liu M, Gingery M, Doulatov SR, Liu Y, Hodes A, Baker S, Davis P, Simmonds M, Churcher C, Mungall K, Quail MA, Preston A, Harvill ET, Maskell DJ, Eiserling FA, Parkhill J, Miller JF. 2004. Genomic and genetic analysis of Bordetella bacteriophages encoding reverse transcriptase-mediated tropism-switching cassettes. J Bacteriol 186:1503–1517. [PubMed][CrossRef]
10. Medhekar B, Miller JF. 2007. Diversity-generating retroelements. Curr Opin Microbiol 10:388–395. [PubMed][CrossRef]
11. Simon DM, Zimmerly S. 2008. A diversity of uncharacterized reverse transcriptases in bacteria. Nucleic Acids Res 36:7219–7229. [PubMed][CrossRef]
12. Minot S, Grunberg S, Wu GD, Lewis JD, Bushman FD. 2012. Hypervariable loci in the human gut virome. Proc Natl Acad Sci U S A 109:3962–3966. [PubMed][CrossRef]
13. Schillinger T, Lisfi M, Chi J, Cullum J, Zingler N. 2012. Analysis of a comprehensive dataset of diversity generating retroelements generated by the program DiGReF. BMC Genomics 13:430. [PubMed][CrossRef]
14. McMahon SA, Miller JL, Lawton JA, Kerkow DE, Hodes A, Marti-Renom MA, Doulatov S, Narayanan E, Sali A, Miller JF, Ghosh P. 2005. The C-type lectin fold as an evolutionary solution for massive sequence variation. Nat Struct Mol Biol 12:886–892. [PubMed][CrossRef]
15. Murphy K. 2012. The Generation of Lymphocyte Antigen Receptors, p. 171. In Janeway's Immunobiology, 8th ed. Garland Science, Taylor & Francis Group, LLC, London and New York.
16. Abbas AK, Lichtman AH, Pillai S. 2012. Lymphocyte Development and Antigen Receptor Gene Rearrangement, p. 186. In Cellular and Molecular Immunology, 7th ed. Elsevier Saunders, Philadelphia, PA.
17. Arambula D, Wong W, Medhekar BA, Guo H, Gingery M, Czornyj E, Liu M, Dey S, Ghosh P, Miller JF. 2013. Surface display of a massively variable lipoprotein by a Legionella diversity-generating retroelement. Proc Natl Acad Sci U S A 110:8212–8217. [PubMed][CrossRef]
18. Stanley NR, Palmer T, Berks BC. 2000. The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli. J Biol Chem 275:11591–11596. [PubMed][CrossRef]
19. Narita S, Tokuda H. 2010. Sorting of bacterial lipoproteins to the outer membrane by the Lol system. Methods Mol Biol 619:117–129. [PubMed][CrossRef]
20. Miller JL, Le Coq J, Hodes A, Barbalat R, Miller JF, Ghosh P. 2008. Selective ligand recognition by a diversity-generating retroelement variable protein. PLoS Biol 6:e131. [PubMed][CrossRef]
21. Dai W, Hodes A, Hui WH, Gingery M, Miller JF, Zhou ZH. 2010. Three-dimensional structure of tropism-switching Bordetella bacteriophage. Proc Natl Acad Sci U S A 107:4347–4352. [PubMed][CrossRef]
22. Le Coq J, Ghosh P. 2011. Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement. Proc Natl Acad Sci U S A 108:14649–14653. [PubMed][CrossRef]
23. Boeke JD, Garfinkel DJ, Styles CA, Fink GR. 1985. Ty elements transpose through an RNA intermediate. Cell 40:491–500. [PubMed][CrossRef]
24. Moran JV, Holmes SE, Naas TP, DeBerardinis RJ, Boeke JD, Kazazian HH, Jr. 1996. High frequency retrotransposition in cultured mammalian cells. Cell 87:917–927. [PubMed][CrossRef]
25. Cousineau B, Smith D, Lawrence-Cavanagh S, Mueller JE, Yang J, Mills D, Manias D, Dunny G, Lambowitz AM, Belfort M. 1998. Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination. Cell 94:451–462. [PubMed][CrossRef]
26. Guo H, Karberg M, Long M, Jones JP, 3rd, Sullenger B, Lambowitz AM. 2000. Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. Science 289:452–457. [PubMed][CrossRef]
27. Alayyoubi M, Guo H, Dey S, Golnazarian T, Brooks GA, Rong A, Miller JF, Ghosh P. 2013. Structure of the essential diversity-generating retroelement protein bAvd and its functionally important interaction with reverse transcriptase. Structure 21:266–276. [PubMed][CrossRef]
28. Luan DD, Korman MH, Jakubczak JL, Eickbush TH. 1993. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72:595–605. [PubMed][CrossRef]
29. Cost GJ, Feng Q, Jacquier A, Boeke JD. 2002. Human L1 element target-primed reverse transcription in vitro. EMBO J 21:5899–5910. [PubMed][CrossRef]
30. Zimmerly S, Guo H, Perlman PS, Lambowitz AM. 1995. Group II intron mobility occurs by target DNA-primed reverse transcription. Cell 82:545–554. [PubMed][CrossRef]
31. Zimmerly S, Guo H, Eskes R, Yang J, Perlman PS, Lambowitz AM. 1995. A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell 83:529–538. [PubMed][CrossRef]
32. Lambowitz AM, Zimmerly S. 2011. Group II introns: mobile ribozymes that invade DNA. Cold Spring Harb Perspect Biol 3:a003616. [PubMed][CrossRef]
33. Kennell JC, Wang H, Lambowitz AM. 1994. The Mauriceville plasmid of Neurospora spp. uses novel mechanisms for initiating reverse transcription in vivo. Mol Cell Biol 14:3094–3107. [PubMed]
34. Chen B, Lambowitz AM. 1997. De novo and DNA primer-mediated initiation of cDNA synthesis by the Mauriceville retroplasmid reverse transcriptase involve recognition of a 3′ CCA sequence. J Mol Biol 271:311–332. [PubMed][CrossRef]
35. George JA, Burke WD, Eickbush TH. 1996. Analysis of the 5′ junctions of R2 insertions with the 28S gene: implications for non-LTR retrotransposition. Genetics 142:853–863. [PubMed]
36. Bibillo A, Eickbush TH. 2002. The reverse transcriptase of the R2 non-LTR retrotransposon: continuous synthesis of cDNA on non-continuous RNA templates. J Mol Biol 316:459–473. [PubMed][CrossRef]
37. Bibillo A, Eickbush TH. 2004. End-to-end template jumping by the reverse transcriptase encoded by the R2 retrotransposon. J Biol Chem 279:14945–14953. [PubMed][CrossRef]
38. Stage DE, Eickbush TH. 2009. Origin of nascent lineages and the mechanisms used to prime second-strand DNA synthesis in the R1 and R2 retrotransposons of Drosophila. Genome Biol 10:R49. [PubMed][CrossRef]
39. Zhuang F, Mastroianni M, White TB, Lambowitz AM. 2009. Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation. Proc Natl Acad Sci U S A 106:18189–18194. [PubMed][CrossRef]
40. White TB, Lambowitz AM. 2012. The retrohoming of linear group II intron RNAs in Drosophila melanogaster occurs by both DNA ligase 4-dependent and -independent mechanisms. PLoS Genet 8:e1002534. [PubMed][CrossRef]
41. 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 Genet 7:e1002414. [PubMed][CrossRef]
42. Crooks GE, Hon G, Chandonia JM, Brenner SE. 2004. WebLogo: a sequence logo generator. Genome Res 14:1188–1190. [PubMed][CrossRef]
43. Schneider TD, Stephens RM. 1990. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18:6097–6100. [PubMed][CrossRef]
44. Dai L, Toor N, Olson R, Keeping A, Zimmerly S. 2003. Database for mobile group II introns. Nucleic Acids Res 31:424–426. [PubMed][CrossRef]
45. Simon DM, Clarke NA, McNeil BA, Johnson I, Pantuso D, Dai L, Chai D, Zimmerly S. 2008. Group II introns in eubacteria and archaea: ORF-less introns and new varieties. RNA 14:1704–1713. [PubMed][CrossRef]
46. Candales MA, Duong A, Hood KS, Li T, Neufeld RA, Sun R, McNeil BA, Wu L, Jarding AM, Zimmerly S. 2012. Database for bacterial group II introns. Nucleic Acids Res 40:D187–D190. [PubMed][CrossRef]
47. Huang H, Chopra R, Verdine GL, Harrison SC. 1998. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282:1669–1675. [PubMed][CrossRef]
48. Kaushik N, Talele TT, Pandey PK, Harris D, Yadav PN, Pandey VN. 2000. Role of glutamine 151 of human immunodeficiency virus type-1 reverse transcriptase in substrate selection as assessed by site-directed mutagenesis. Biochemistry 39:2912–2920. [PubMed][CrossRef]
49. Sarafianos SG, Hughes SH, Arnold E. 2004. Designing anti-AIDS drugs targeting the major mechanism of HIV-1 RT resistance to nucleoside analog drugs. Int J Biochem Cell Biol 36:1706–1715. [PubMed][CrossRef]
50. Boyer PL, Sarafianos SG, Clark PK, Arnold E, Hughes SH. 2006. Why do HIV-1 and HIV-2 use different pathways to develop AZT resistance? PLoS Pathog 2:e10. [PubMed][CrossRef]
51. Mansky LM, Temin HM. 1995. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol 69:5087–5094. [PubMed]
52. Abram ME, Ferris AL, Shao W, Alvord WG, Hughes SH. 2010. Nature, position, and frequency of mutations made in a single cycle of HIV-1 replication. J Virol 84:9864–9878. [PubMed][CrossRef]
53. Schillinger T, Zingler N. 2012. The low incidence of diversity-generating retroelements in sequenced genomes. Mob Genet Elements 2:287–291. [PubMed][CrossRef]
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Abstract:

Diversity-generating retroelements (DGRs) are DNA diversification machines found in diverse bacterial and bacteriophage genomes that accelerate the evolution of ligand–receptor interactions. Diversification results from a unidirectional transfer of sequence information from an invariant template repeat (TR) to a variable repeat (VR) located in a protein-encoding gene. Information transfer is coupled to site-specific mutagenesis in a process called mutagenic homing, which occurs through an RNA intermediate and is catalyzed by a unique, DGR-encoded reverse transcriptase that converts adenine residues in the TR into random nucleotides in the VR. In the prototype DGR found in the bacteriophage BPP-1, the variable protein Mtd is responsible for phage receptor recognition. VR diversification enables progeny phage to switch tropism, accelerating their adaptation to changes in sequence or availability of host cell-surface molecules for infection. Since their discovery, hundreds of DGRs have been identified, and their functions are just beginning to be understood. VR-encoded residues of many DGR-diversified proteins are displayed in the context of a C-type lectin fold, although other scaffolds, including the immunoglobulin fold, may also be used. DGR homing is postulated to occur through a specialized target DNA-primed reverse transcription mechanism that allows repeated rounds of diversification and selection, and the ability to engineer DGRs to target heterologous genes suggests applications for bioengineering. This chapter provides a comprehensive review of our current understanding of this newly discovered family of beneficial retroelements.

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FIGURE 1

BPP-1 phage and its diversity-generating retroelement (DGR). The BPP-1 genome is represented in the prophage form flanked by a duplication of the His-tRNA gene formed during integration. Functional assignments for most gene clusters are indicated, along with the -like repressor and the DGR cassette. Schematic representation of the DGR cassette and its function in phage tropism switching. The cassette contains three genes (, , and ) and two 134-bp repeats (template and variable repeats, or TR and VR, respectively). The VR is located at the 3′ end of the gene, which encodes the distal tail fiber protein responsible for receptor recognition. Located at the 3′ ends of the VR and TR are IMH (initiation of mutagenic homing) and IMH* elements, respectively, in addition to a GC-rich element. Phage tropism switching occurs through DGR-mediated mutagenic homing, in which TR sequence information is transferred to the VR with adenine residues in the TR appearing as random nucleotides in the VR. Shown on the bottom are electron micrographs of the BPP-1 phage; globular structures at the distal ends of tail fibers are Mtd trimers (two per fiber). Comparison of the BPP-1 TR and VR. The TR and VR sequences are shown in bold. The VR variable positions and the corresponding adenine residues in the TR are shown in red. IMH, IMH*, and GC-rich elements are also indicated. There are 23 adenines in the TR, which can theoretically generate ∼10 different DNA sequences, or ∼10 different peptides. (Adapted from 7 and 9 .) doi:10.1128/microbiolspec.MDNA3-0029-2014.f1

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0029-2014
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FIGURE 2

Diversification of a surface-displayed lipoprotein by a DGR. The strain Corby DGR is encoded on a genomic island that differs in G+C content from the rest of the genome. The VR sequences at the 3′ end of the diversified locus, , are flanked by tandem hairpin/cruciform structures that are essential for efficient homing. The TR contains 43 adenine residues, which can create a potential repertoire of 10 different VR DNA sequences. LdtA contains atypical TAT (win rginine ransport) and Lpp (lipobox, lipid modification) signals at the N terminus. Cellular localization studies demonstrated that LdtA is exported through the inner membrane via the TAT pathway, lipid modified, and anchored on the outer surface of the outer membrane via an Lpp-like lipoprotein processing pathway. VR-encoded residues are surface displayed by a C-terminal CLec fold. (Adapted from 17 .) doi:10.1128/microbiolspec.MDNA3-0029-2014.f2

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0029-2014
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FIGURE 3

The CLec fold as a scaffold for display of DGR-generated protein diversity. Left: The BPP-1 Mtd protein forms a pyramid-shaped homotrimer with VR-encoded residues exposed on the bottom surface. Right: An Mtd monomer containing β-prism, β-sandwich, and VR-encoded CLec domains, from the N to the C terminus. Backbone structures of the VR regions of five Mtd variants with different ligand specificities are shown. Despite side-chain variations in diversified VR residues, the backbone structures are nearly superimposable. Comparison of the CLec VR regions of BPP-1 Mtd and a variable protein, TvpA. For the Mtd VR, the β2β3 loop of a second monomer is also shown (blue). Superposition of the VR regions of BPP-1 Mtd (light orange) and TvpA (blue). Interaction of an Mtd homotrimer with the receptor protein pertactin. See text for details. (Adapted from 14 , 20 , and 22 .) doi:10.1128/microbiolspec.MDNA3-0029-2014.f3

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0029-2014
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FIGURE 4

The TPRT model of BPP-1 DGR-mediated mutagenic homing. Mutagenic homing occurs through a TR RNA intermediate and is RecA independent, similar to group II intron homing. A marker co-conversion assay mapped the cDNA transfer boundary to a narrow region within the GC-rich element at the 3′ end, which may represent 3′ cDNA integration site(s). The marker transfer boundary at the 5′ end was more heterogeneous. A target DNA-primed reverse transcription model, similar to that of group II intron homing, has been proposed to explain these observations. The DNA target site was hypothesized to be nicked within the GC-rich element, with the exposed 3′-hydroxyl group serving as a primer for adenine-specific error-prone reverse transcription of the TR RNA. Integration of cDNA products at the 5′ end requires short stretches of homology between the VR and the cDNA and may occur through strand displacement or template switching followed by break repair. Subsequent DNA replication would then create progeny genomes with mutagenized variable regions. Deletion of the VR sequence upstream of GC and IMH elements appeared to block 5′ cDNA integration but not 3′ cDNA integration, as analyzed by PCR with primer sets 1/4 and 2/3, respectively. Sequence analysis showed adenine mutagenesis in PCR products generated with primers 2&3. 5′ cDNA integration in ΔVR1-99 was restored by inserting a 50 bp sequence, which is homologous to the region upstream of the deletion junction, in the TR. (Adapted from reference 7 ) doi:10.1128/microbiolspec.MDNA3-0029-2014.f4

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0029-2014
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FIGURE 5

Role of a DNA secondary structure in DGR target recognition. A DNA hairpin/cruciform structure downstream of the VR is required for BPP-1 DGR target recognition. The wild-type (WT) structure contains an 8-bp GC-rich stem and a 4-nt GAAA loop and is located 4 bp downstream of the VR. Mutating the 3′ half of the stem (StMut) dramatically reduced DGR mutagenic homing and phage tropism switching (not shown), while complementary changes to the 5′ half of the stem (StRev) restored DGR activity in both assays. PCR-based DGR homing assays with sequence-tagged TRs and VRs flanked by WT or mutant stem sequences. Shown on the right is a diagram of the PCR assay. Green represents the tag sequence transferred from the TR to the VR. P1 to P4 are primers annealing to the tag or flanking regions. Similar DNA structures are found at analogous positions in a number of other phage (two shown) and bacterial (one shown) DGRs. The phage stems are GC rich and range from 7 to 10 bp, and loops have a conserved 4-nt sequence, G(A/G)NA. The Corby DGR has a more complex tandem structure that is required for homing. BPP-1 DGR target recognition at the 3′ end is both sequence and structure dependent, requiring GC, IMH, and a hairpin/cruciform structure. Target recognition at the 5′ end is homology mediated. By inserting a gene of interest (GOI) upstream of GC, IMH, and the DNA structure, the heterologous gene can be diversified by the BPP-1 DGR through appropriate engineering of the TR. (Adapted from 41 .) doi:10.1128/microbiolspec.MDNA3-0029-2014.f5

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0029-2014
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FIGURE 6

Avd and Brt. Left: The BPP-1 Avd protein forms a homopentameric structure, with each monomer containing four helices running up and down (side view). The pentamer is highly positively charged (top view; blue, positively charged; red, negatively charged). Right: Amino acid residues on the side, top, and bottom of the Avd pentamer that were tested for Avd–Brt binding and/or DGR homing ( 27 ). DGR RT domains and the sequence logo of its highly conserved domain R4. R1 to R7 are conserved sequence blocks found in the finger and palm domains of retroviral RTs, such as human immunodeficiency virus type 1 (HIV-1) RT (bottom). DGR RTs contain sequence insertions between R2 and R3 (R2a), and between R3 and R4 (R3a), as well as divergent N and C termini. They do not contain the thumb (Th) and RNase H (RH) domains that are found in HIV-1 RT. The domain R4 sequence logo of 155 DGR RTs was generated by Schillinger using WebLogo ( 13 , 42 , 43 ). Comparison with the domain R4 sequence logo that we generated from 93 bacterial group II intron RTs (group II intron database: http://webapps2.ucalgary.ca/∼groupii/orf/orfalignment.html; [ 44 , 45 , 46 ]), which are most closely related to DGR RTs, showed several characteristic differences, including the two highly conserved positions labeled with an asterisk (*). Also included for comparison is the corresponding amino acid sequence block of HIV-1 RT (strain BRU; GenBank accession no. K02013). The glutamine residue at position 151, which plays a role in nucleotide and template preference during reverse transcription, is highlighted in blue. (Adapted from 13 .) doi:10.1128/microbiolspec.MDNA3-0029-2014.f6

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