General features and properties of insertion sequence elements

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The HUH Catalytic site

Mechanism and Overall Protein Architecture.

Historically, bacteriophage fX174 protein A (gpA) was the first identified HUH superfamily member (see (Kornberg & Baker, 1992)) although, surprisingly, no structural information is available.

Many related proteins subsequently identified by bioinformatics (Ilyina & Koonin, 1992, Koonin & Ilyina, 1993) included proteins involved in catalysis of viral and plasmid rolling circle replication (RCR), conjugative plasmid transfer, and DNA transposition (Kapitonov & Jurka, 2001, Garcillan-Barcia, et al., 2002, Ronning, et al., 2005, Ton-Hoang, et al., 2005, Toleman, et al., 2006, Garcillan-Barcia, et al., 2009). They all carry conserved protein motifs, including the "HUH" motif composed of two histidine (H) residues separated by a bulky hydrophobic (U) residue, and the Y-motif containing either one or two tyrosine (Tyr) residues (found in Y1 and Y2 enzymes respectively).

Y1 HUH enzymes (Fig 1.41.1) include Rep proteins of some plasmids with ssDNA replication intermediates (such as pUB110 (Gruss & Ehrlich, 1989), a wide range of eukaryotic viruses (Rosario, et al., 2012), most conjugative plasmid relaxases (de la Cruz, et al., 2010, Guglielmini, et al., 2011), ISCR (insertion sequences related to IS91(Toleman, et al., 2006)) and IS200/IS605 insertion sequence family transposases (Ronning, et al., 2005, Ton-Hoang, et al., 2005). Y2 enzymes include fX174 gpA itself, Rep proteins of other isometric ssDNA and dsDNA phages (e.g. phage P2 (Odegrip & Haggard-Ljungquist, 2001)), some cyanobacterial and archaeal plasmids and parvoviruses (e.g. adeno-associated virus, AAV) as well as transposases of the IS91 and helitron families (Kapitonov & Jurka, 2001), and MOBF family plasmid relaxases. In some cases, both Y residues are mechanistically important while for others, only one of the pair appears to be essential.

HUH enzymes use a unique mechanism for catalysing ssDNA breakage and joining. The active site tyrosine creates a 5'-phosphotyrosine intermediate and a free 3'-OH at the cleavage site (Fig 1.41.1). The 3'-OH can be used for different tasks. The most obvious is to prime replication, as observed for HUH domains in single-stranded phage Rep proteins, RCR plasmids and conjugative relaxases. The 3'-OH group can also act as the nucleophile for strand transfer to resolve the phosphotyrosine intermediate in the termination step of RCR replication, conjugative transfer and transposition.

The HUH enzyme cleavage polarity is inverse to that of the tyrosine recombinases, which make 3' phosphotyrosine intermediates and generate free 5'-OH groups that cannot be used as replication primers (Grindley, et al., 2006). HUH enzymes also require a divalent metal ion to facilitate cleavage by localizing and polarising the scissile phosphodiester bond in contrast to the cofactor-independent tyrosine recombinases. Depending on the enzyme, Mg2+, Mn2+ or other divalent metal ions can be used in vitro (Datta, et al., 2003, Larkin, et al., 2005, Boer, et al., 2006, Boer, et al., 2009, Hickman, et al., 2010, Edwards, et al., 2013). It is presumed that Mg2+ or Mn2+ are the physiological cofactors. The HUH histidine pair provides two of the three ligands necessary for metal ion coordination (Fig 1.41.1). The location and identity of the third, invariably polar (Glu, Asp, His or Gln) residue varies across the superfamily.

3D structures of several Rep and relaxase HUH domains with and without bound DNA are available e.g. (Datta, et al., 2003, Guasch, et al., 2003, Larkin, et al., 2003, Hickman, et al., 2004, Boer, et al., 2006, Boer, et al., 2009, Messing, et al., 2012). The order of HUH and Y motifs varies in the primary sequence: in the Relaxase group, the Y-motif is upstream of the HUH-motif whereas in the Rep group it is downstream (Fig1.41.1). This "circular permutation" (Koonin & Ilyina, 1993, Dyda & Hickman, 2003, Guasch, et al., 2003) changes the domain topology. Nevertheless, the three-dimensional constellation of active site residues is virtually identical across the superfamily.

Given the diverse HUH protein functions, it is not surprising that other domains are often appended to the HUH domain (Fig1.41.1). These are often of unknown function but, ATP dependent helicase, zinc binding, primase and multimerisation domains are recurring themes (Petit, et al., 1998, Bruand & Ehrlich, 2000, Odegrip, et al., 2000, Kapitonov & Jurka, 2001, Chang, et al., 2002, Hickman, et al., 2004, Clerot & Bernardi, 2006). For example, the ssDNA substrates needed by HUH enzymes can be generated by a dedicated DNA helicase domain C-terminal to the HUH domain (Im & Muzyczka, 1990, Brister & Muzyczka, 1999, Kapitonov & Jurka, 2001, Clerot & Bernardi, 2006) or alternatively by recruitment of a host helicase (Petit, et al., 1998, Bruand & Ehrlich, 2000, Odegrip, et al., 2000, Chang, et al., 2002). RCR processes use 3'-5'helicase activity acting on the template strand to facilitate DNA unwinding at the replication fork while in conjugation, helicases (as part of the relaxase) are transported into the recipient cell and track 5' to 3' on the transported ssDNA.

DNA Recognition

Many HUH nucleases recognize and bind DNA hairpin structures with cleavage sites located within the hairpin or in the ssDNA on the 5' or 3' side of the stem. The crucial role of hairpins has been firmly established in many systems including plasmid conjugation, eukaryotic viral and plasmid replication and transposition (Orozco & Hanley-Bowdoin, 1996, Brister & Muzyczka, 2000, Ronning, et al., 2005, Ton-Hoang, et al., 2005, Boer, et al., 2006, Messing, et al., 2012, Ton-Hoang, et al., 2012). In other systems, palindromic sequences that can form DNA hairpins are present near the probable HUH nuclease cleavage sites [Feschotte, 2001] (del Solar, et al., 1998). Such hairpins can be formed in vivo under a number of physiological conditions (see (Bikard, et al., 2011)).

Structural studies revealed that small DNA hairpins can be recognized in several different ways: sequence-specific recognition of the dsDNA stem; structure-specific recognition of irregularities in the stem; or sequence-specific recognition of the hairpin loop (Guasch, et al., 2003, Hickman, et al., 2004, Ronning, et al., 2005, Hickman, et al., 2010, Messing, et al., 2012, Edwards, et al., 2013).

The hairpin-flanking DNA - in many cases in single-stranded form - is also often important for recognition. Relaxases, for example, make extensive contacts with the bases extending between the hairpin and cleavage site (Guasch, et al., 2003, Larkin, et al., 2003, Edwards, et al., 2013), and for a relative of IS200/IS605 transposases, TnpAREP, nucleotides on the 5' side of the hairpin are crucial for binding and sequence-specific recognition (Messing, et al., 2012). Other family members (Hickman, et al., 2004, Ruiz-Maso, et al., 2007), have more complex binding modes.

HUH enzymes as transposases

Transposases of members of the IS200/IS605(Ton-Hoang, et al., 2005), IS91 (Mendiola, et al., 1994) and ISCR (Toleman, et al., 2006) insertion sequence families and the eukaryotic helitrons (Kapitonov & Jurka, 2001) are also HUH enzymes. Those IS200/IS605 family are the best understood.

IS200/IS605 family

IS200/IS605 family transposases are single domain proteins with only the essential HUH motif and a single catalytic Tyr (Y1 transposases, Fig 1.41.1). Both TnpAIS608 and TnpAISDra2 are obligatory dimers and the active sites are believed to adopt two functionally important conformations, one in which each is composed of the HUH motif from one monomer and the Tyr residue carried by an alpha-helix (aD) from the other (trans configuration), and the other in which both motifs are contributed by the same monomer (cis configuration). Only the former has been observed crystallographically.

Similar proteins are sometimes found associated with repeated Extragenic Palindromes (REP sequences whose hairpin structures resemble the ends of IS200/IS605 family members.

RCR transposons: IS91, ISCR and Helitron families

The earliest identified HUH domain transposases were those of the IS91 family (Garcillan-Barcia, et al., 2002) and are significantly larger than Y1 transposases (Fig 1.41.1), carry a Y2 motif and include an N-terminal zinc binding motif and additional domains of yet unidentified function.

A group of related elements, the ISCRs often associated with a variety of antibiotic resistance genes (see (Toleman, et al., 2006)) carry an orf (the CR or common region) resembling IS91 family transposases but with only a single Tyr (Fig 1.41.1). In addition, eukaryotic relatives, the Helitrons, have been identified by bioinformatic approaches.

    References :
  • Bikard D, Loot C, Baharoglu Z & Mazel D (2011) Folded DNA in action: hairpin formation and biological functions in prokaryotes. Microbiol Mol Biol Rev 74: 570-588.
  • Boer DR, Ruiz-Maso JA, Lopez-Blanco JR, et al. (2009) Plasmid replication initiator RepB forms a hexamer reminiscent of ring helicases and has mobile nuclease domains. EMBO J 28: 1666-1678.
  • Boer R, Russi S, Guasch A, et al. (2006) Unveiling the molecular mechanism of a conjugative relaxase: The structure of TrwC complexed with a 27-mer DNA comprising the recognition hairpin and the cleavage site. J Mol Biol 358: 857-869.
  • Brister JR & Muzyczka N (1999) Rep-mediated nicking of the adeno-associated virus origin requires two biochemical activities, DNA helicase activity and transesterification. J Virol 73: 9325-9336.
  • Brister JR & Muzyczka N (2000) Mechanism of Rep-mediated adeno-associated virus origin nicking. J Virol 74: 7762-7771.
  • Bruand C & Ehrlich SD (2000) UvrD-dependent replication of rolling-circle plasmids in Escherichia coli. Mol Microbiol 35: 204-210.
  • Chang TL, Naqvi A, Anand SP, Kramer MG, Munshi R & Khan SA (2002) Biochemical characterization of the Staphylococcus aureus PcrA helicase and its role in plasmid rolling circle replication. J Biol Chem 277: 45880-45886.
  • Clerot D & Bernardi F (2006) DNA helicase activity is associated with the replication initiator protein rep of tomato yellow leaf curl geminivirus. J Virol 80: 11322-11330.
  • Datta S, Larkin C & Schildbach JF (2003) Structural insights into single-stranded DNA binding and cleavage by F factor TraI. Structure 11: 1369-1379.
  • de la Cruz F, Frost LS, Meyer RJ & Zechner EL (2010) Conjugative DNA metabolism in Gram-negative bacteria. FEMS Microbiol Rev 34: 18-40.
  • del Solar G, Giraldo R, Ruiz-Echevarria MJ, Espinosa M & Diaz-Orejas R (1998) Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev 62: 434-464.
  • Dyda F & Hickman AB (2003) A mob of reps. Structure (Camb) 11: 1310-1311.
  • Edwards JS, Betts L, Frazier ML, et al. (2013) Molecular basis of antibiotic multiresistance transfer in Staphylococcus aureus. Proc Natl Acad Sci U S A 110: 2804-2809.
  • Garcillan-Barcia MP, Francia MV & de la Cruz F (2009) The diversity of conjugative relaxases and its application in plasmid classification.FEMS Microbiol Rev 33: 657-687.
  • Garcillan-Barcia MP, Bernales I, Mendiola MV & De la Cruz F (2002) IS91 rolling circle transposition. Mobile DNA, Vol. II (Craig NL, Craigie R, Gellert M & Lambowitz A, eds.), pp. 891-904. ASM press, Washington DC.
  • Grindley ND, Whiteson KL & Rice PA (2006) Mechanisms of site-specific recombination. Annu Rev Biochem 75: 567-605.
  • Gruss A & Ehrlich SD (1989) The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev 53: 231-241.
  • Guasch A, Lucas M, Moncalian G, et al. (2003) Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC. Nat Struct Biol 10: 1002-1010.
  • Guglielmini J, Quintais L, Garcillan-Barcia MP, de la Cruz F & Rocha EP (2011) The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation. PLoS Genet 7: e1002222.
  • Hickman AB, Ronning DR, Perez ZN, Kotin RM & Dyda F (2004) The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces. Mol Cell 13: 403-414.
  • Hickman AB, James JA, Barabas O, et al. (2010) DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans. Embo J 29: 3840-3852.
  • Ilyina TV & Koonin EV (1992) Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic.Acids.Res. 20: 3279-3285.
  • Im DS & Muzyczka N (1990) The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 61: 447-457.
  • Kapitonov VV & Jurka J (2001) Rolling-circle transposons in eukaryotes. Proc Natl Acad Sci U S A 98: 8714-8719.
  • Koonin EV & Ilyina TV (1993) Computer-assisted dissection of rolling circle DNA replication. Biosystems 30: 241-268.
  • Kornberg A & Baker TA (1992) DNA Replication. W.H.Freeman, New York.
  • Larkin C, Datta S, Nezami A, Dohm JA & Schildbach JF (2003) Crystallization and preliminary X-ray characterization of the relaxase domain of F factor TraI. Acta Crystallogr D Biol Crystallogr 59: 1514-1516.
  • Larkin C, Datta S, Harley MJ, Anderson BJ, Ebie A, Hargreaves V & Schildbach JF (2005) Inter- and intramolecular determinants of the specificity of single-stranded DNA binding and cleavage by the F factor relaxase. Structure 13: 1533-1544.
  • Mendiola MV, Bernales I & de la Cruz F (1994) Differential roles of the transposon termini in IS91 transposition. Proc.Natl.Acad.Sci.U.S.A. 91: 1922-1926.
  • Messing SA, Ton-Hoang B, Hickman AB, et al.(2012) The processing of repetitive extragenic palindromes: the structure of a repetitive extragenic palindrome bound to its associated nuclease. Nucleic Acids Res 40: 9964-9979.
  • Odegrip R & Haggard-Ljungquist E (2001) The two active-site tyrosine residues of the a protein play non-equivalent roles during initiation of rolling circle replication of bacteriophage p2. J Mol Biol 308: 147-163.
  • Odegrip R, Schoen S, Haggard-Ljungquist E, Park K & Chattoraj DK (2000) The interaction of bacteriophage P2 B protein with Escherichia coli DnaB helicase. J Virol 74: 4057-4063.
  • Orozco BM & Hanley-Bowdoin L (1996) A DNA structure is required for geminivirus replication origin function. J Virol 70: 148-158.
  • Petit MA, Dervyn E, Rose M, Entian KD, McGovern S, Ehrlich SD & Bruand C (1998) PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling-circle replication. Mol Microbiol 29: 261-273.
  • Ronning DR, Guynet C, Ton-Hoang B, Perez ZN, Ghirlando R, Chandler M & Dyda F (2005) Active site sharing and subterminal hairpin recognition in a new class of DNA transposases. Mol Cell 20: 143-154.
  • Rosario K, Duffy S & Breitbart M (2012) A field guide to eukaryotic circular single-stranded DNA viruses: insights gained from metagenomics. Arch Virol 157: 1851-1871.
  • Ruiz-Maso JA, Lurz R, Espinosa M & del Solar G (2007) Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication. Nucleic Acids Res 35: 1230-1244.
  • Toleman MA, Bennett PM & Walsh TR (2006) ISCR Elements: Novel Gene-Capturing Systems of the 21st Century? Microbiol Mol Biol Rev 70: 296-316.
  • Ton-Hoang B, Guynet C, Ronning DR, Cointin-Marty B, Dyda F & Chandler M (2005) Transposition of ISHp608, member of an unusual family of bacterial insertion sequences. Embo J 24: 3325-3338.
  • Ton-Hoang B, Siguier P, Quentin Y, Onillon S, Marty B, Fichant G & Chandler M (2012) Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences. Nucleic Acids Res 40: 3596-3609.