Dreyfus DH .

T32 AI007365 NIAID NIH HHS , T32 GM007288 NIGMS NIH HHS , T32-AI07365 NIAID NIH HHS , T32-GM07288 NIGMS NIH HHS

	Figure 1. Mechanism of Tc element transposition, herpes recombination and V(D)J recombination suggests homologous adaptation of a primordial DDE recombinase.Recombination sites of all of these DNA elements are adjacent to a sequence containing a sequence resembling the V(D)J heptamer (bold type) and nonamer sequences (underlined). Nonamer sequences are often spaced at 12 or 23 nucleotide intervals to facilitate DNA bending. Sequences shown from top to bottom: invertebrate Tc elements Tc1, Tc3, EBV terminal repeat sequences, herpes simplex recombination sites, V(D)J RSS and transib transposon termini (Tsib) most closely related to V(D)J RSS among tranposons.
	Figure 2. The complete sequence of an EBV terminal repeat defined by the SauIII restriction enzyme is shown with putative V(D)J-like regions and Sp1 transcription factor binding sites identified.Location of V(D)J-like sequences in EBV terminal repeats adjacent to experimentally confirmed Sp1 protein binding sites. Specific protein complexes distinct from Sp1 are also evident on the EBV V(D)J-like sequences shown , and these sequences undergo anomalous migration on native polyacrylamide gels typical of bent DNA similar to the V(D)J RSS and to transposon termini (unpublished observations).
	Figure 3. The “big bang” or “RAG-transposon insertion model” of the origins of the acquired immune system.As shown , a transposon inserted at a site in the genome denoted Site A can transcribe an mRNA encoding a bi-molecular transposase consisting of RAG-1 and RAG-2 like proteins from promoters in flanking sequences. Expression of the transposase then can excise a transposon from site A or another site (large arrows) at the transposon termini and insert the transposon and another site termed site B with an immunoglobulin or T-cell receptor gene. Subsequent excision of V(D)J RSS that resemble transposon termini results in circular episomes and repaired empty sites in immunoglobulin and T cell receptor genes. Multiple cycles of RAG transposon insertion and excision from primordial immunoglobulin and T-cell receptor like genes and insertion of the RAG transposon at other sites in the genome such as the current RAG locus with subsequent gene amplification of the immunoglobulin and T cell receptor gene families could result in the current structure of these genetic loci.
	Figure 4. The Co-regulatory model including initial insertion of a primordial herpes virus recombinase (proto-RAG-1 denoted pR1) adjacent to a pre-existing RAG-2 like protein (denoted pR2) is shown.As shown, insertion of a herpes virus episome or linear genome adjacent to a RAG-2 like gene would provide a master co-regulated RAG-2/RAG-2 locus acting subsequently through co-evolving slave RSS sites in immunoglobulin or T-cell receptor genes. Co-evolving slave RSS could arise either from additional herpes or transposon insertions and gene duplication events or from co-evolution of endogenous sequences with some similarity to transposon or herpes virus termini in other genes such as those encoding B- and T-lymphocyte receptors (Figure 1). In contrast to the “RAG transposon” model, the co-regulatory model does not require the existence of a composite RAG-1/RAG-2 transposase or transposon and can also account for the experimental structure of the current RAG-1/RAG-2-like genes in the sea urchin and other deuterostomes that do not undergo V(D)J recombination.
	Figure 5. Shared somatic regulation between the EBV DBP BALF-2 protein gene and RAG.As shown, a 200 NT 5′ region immediately adjacent to EBV BALF-2 ORF AUG start codon contains putative regulatory sequences for BZLF-1/AP-1 (denoted with a single asterisk), CREB (denoted with a double asterisk**), and SP1 (denoted with a triple asterisk***). These putative regulatory sequences are enclosed in boxes in the figure and include sequences recognized by the EBV encoded BZLF-1 regulatory protein (also termed ZEBRA protein). BZLF-1 sites also are also functional as sites for the endogenous regulatory factor AP-1 as discussed in the text. BZLF-1 regulated sites from other EBV genes ZIIIA, B, and ZRE1,2,3,5 are shown in comparison to a consensus AP-1 site in the lower portion of the figure. Similarly, in the lower portion of the figure the putative binding site for CREB is shown in the BALF-2 minimal promoter in comparison to Col8, a cAMP response element shown to bind CREB1 cAMP site binding protein with high affinity and ZII, a site in the BZLF-1 promoter shown experimentally to respond to cAMP. Also in the lower portion of the figure, a site in the BALF-2 minimal promoter matching the Sp1 transcription factor consensus is shown, similar but not identical to Sp1 binding sites confirmed to exist in the EBV terminal repeats (Figure 2).
	Figure 6. The hypothesis that a herpes DBP-like protein and RAG-1 protein have a modular architecture with structural and functional homology of functions is presented.Primordial RAG-1 protein (denoted pR1) has a carboxyl region structurally similar to a transib transposase (denoted T for Transib-like region structure #1), but extra amino terminus protein sequences that may be derived from another protein family (denoted N). Herpes DBP are magnesium dependent recombinases are also modular proteins with an amino terminal regulatory region (denoted N), and a carboxyl terminus that binds to DNA. The RAG-1 protein currently requires a physical association with the RAG-2 protein for recombinase activity in vivo, but may have initially exhibited recombinase properties without RAG-2 analogous to the DBP. As discussed in the text, primordial RAG-2 protein (denoted pR2) may initially have blocked the recombinase functions of pR1 but exposed immunologic determinants essential to herpes virus immunity since the DBP are a major herpes virus antigen. Both Herpes DBP and RAG-1 also require an association with host cell factors such as DNApk and ku shared with the RAG proteins for viral recombinase activity in vivo as discussed in more detail in the text.
	Figure 7. Summary of functional correlates between RAG-1 and herpes DBP (asterix indicates observations novel to this work, other observations presented previously).These functional correlates are consistent with a homologous descent of both proteins from a common precursor recombinase although analogous convergence of functional properties cannot be excluded.
	Figure 8. Conserved Functional DDE residues between transposases, RAG proteins and herpes DBP.Despite primary “low information content” amino acid sequence divergence of intervening sequences, RAG-1 proteins encode a “high information content” absolutely conserved E residue adjacent to a conserved alpha helix in the extreme carboxyl terminus of the protein shared with prokaryotic transposons (Tn5 and Tn10). This functionally conserved residue is required for RAG-1 magnesium ion binding and protein function. Similarly, despite primary amino acid sequence divergence of intervening regions all herpes DBP encode a conserved D/E residue adjacent to a conserved alpha helix in the DNA-binding carboxyl terminus of the protein. These high information content similarities are consistent with and support descent of both proteins from a common precursor recombinase.
	Figure 9. Putative magnesium ion binding regions of the DBP can be localized adjacent to the DNA binding groove of the ICP-8 protein structure.The partial crystal structure of herpes simplex DBP ICP-8 is shown with experimentally determined DNA binding groove shown, while experimentally determined structures of RAG proteins and other herpes DBP are not solved currently. A black double arrow illustrates the experimentally determined DNA binding groove of ICP-8, while a green arrow indicates the hypothetical position of a bound magnesium ion in ICP-8 as localized by conserved blocks of D and E residues shared with RAG-1 in regions of ICP-8 (Figure 8). This alignment shows that the predicted Mg binding site geometry of ICP-8 is in proximity to the bound DNA as in other structurally characterized DDE enzymes such as RISC. These structural similarities are consistent with and support descent of DBP and RISC proteins from a common precursor DDE recombinase.
	Figure 10. Possible scenarios for origins of V(D)J recombination in the absence of a “RAG Transposon.”The co-regulatory hypothesis presented in this work cannot exclude the possibility that a transib transposon-like element inserted directly into the current RAG locus adjacent to a primordial RAG-2 gene and a conveniently located independent N terminal-like protein with sequences somatically regulated in immune cells already present at the site as shown in the top scenario (Scenario 1). However, this scenario would require several independent coincidences of adjacent N protein and regulatory sequences adjacent to proto RAG2 not found experimentally. Scenario 1 also provides no explanation for the continued presence of the RAG-1/RAG-2-like locus in the modern sea urchin genome in the absence of any known function or slective advantage. In a more probable scenario shown in the bottom panel a herpes-like episome already containing N terminal protein sequences and cis-linked somatic regulatory sequences inserted adjacent to a primordial RAG-2 protein to generate the current RAG site (Scenario 2). After the initial generation of the RAG site in either scenario, the initial selective benefit of the RAG locus may have been to provide immunity to subsequent herpes virus infection rather than V(D)J recombination for an undetermined interval of time during which herpes and RAG protein primary sequences diverged, and this locus may still provide some partial immunity until the present time to conserved “high-information content” regions of the herpes recombinase that cannot diverge due to functional constraints . After the primordial herpes virus lineage had diverged sufficiently in primary sequence to permit re-infection of the primordial deuterostome host with herpes-like pathogens, resumption of the herpes-host arms race would continue until the present.

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