BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/8 The secondary structure predictions showed that the Rad52 superfamily proteins adopt a structure with inter- spersed α-helices and β-strands (Fig. 5). Additionally, fold predictions using 3DPSSM (E-value=.0085, correspond- ing to a 90% confidence in the prediction) and the hybrid fold method (Z-score = 19.5) predicted the presence of a potential Helix-hairpin-Helix (HhH) fold in members of the RAD52 superfamily. The HhH domain is a small nu- cleic acid-binding module comprised of two helices joined by a central loop (hairpin), which functions as the DNA-binding moiety of numerous repair and recombina- tion proteins[27,28]. Two HhH modules are predicted in the core conserved domain of the RAD52 family, the first one bounded by the predicted helices 2 and 3, and the sec- ond one bounded by helices 5 and 6 (Fig. 5). Although these predicted HhH modules are very divergent in se- quence from the typical versions, the hairpin in both HhH modules of the RAD52 family proteins is bounded by small residues, typically glycine; this conforms to the sig- nature motif characteristic of the classical HhH modules [28,29]. However, in the case of the RAD52 superfamily the predicted HhH modules appear to have been welded into a large globular superstructure that maintained its ev- olutionary distinctness over time. The conservation pat- tern and predicted structural elements of the RAD52 superfamily are distinct from those predicted for the ERF and RecT/Redβ superfamilies (Fig 1, 3, 5), supporting the lack of a direct evolutionary relationship between these proteins. The RAD52 superfamily shows a sporadic phyletic distri- bution, and even in the crown-group eukaryotes, might have been secondarily lost in certain lineages, such as plants, nematodes and insects. The sporadic distribution of this family among phylogenetically distant bacteria, along with its presence in several prophages, suggests that, like the RecT/Redβ and ERF superfamilies, at least the bac- terial RAD52-proteins might be of predominantly phage origin. The core of the eukaryotic recombination system appears to have been inherited from the system present in the common ancestor shared with the archaea [29]. How- ever, RAD52 is thus far absent in all archaeal genomes and is restricted to a single orthologous group in the eukaryo- tes [29]. Thus, it appears plausible that eukaryotic RAD52 was ultimately derived through lateral transfer either from a bacterial genome or directly from a viral source, at a point at least predating the divergence of the crown group eukaryotes and Entamoeba. Contextual information from gene neighborhoods pro- vides details regarding functional interactions of the SSAPs with DNA recombination pathways The clustering of functionally related genes in prokaryotic genomes into co-transcribed and co-regulated units, oper- ons, often allows functional assignments through the principle of 'guilt by association' [30–32]. Generally, genes whose products physically interact to form a com- plex or are involved in successive steps in a biochemical pathway form operons that are conserved over large evo- lutionary distances [30]. On previous occasions, we have used gene neighborhoods or operons to predict novel DNA repair complexes and their components [33]. Ac- cordingly, a similar approach was applied to the three families of SSAPs (RecT/Redβ, ERF, Rad52), to shed light on their functional links. Notably, the genes encoding the three evolutionarily dis- tinct SSAPs co-occurred with similar sets of DNA repair/re- combination-related proteins (Figs. 2,4). In at least one case, each of them was found adjacent to the gene for the single-strand-binding protein (SSB), an OB-fold protein that binds ssDNA (Figs. 2,4). This association ties in with the function of the SSAPs in single-strand annealing, sug- gesting that they closely interact with SSB. It has been sug- gested in the case of RecT that it may compete with SSB for binding single strand overhangs and thereby make them available for the annealing process [3]. Similar interac- tions between other SSAPs and SSB, that probably coats the ssDNA generated by nucleases, appear likely. Genes for SSAPs from all the 3 distinct superfamilies may also occur adjacent to or in the vicinity of genes encoding nu- cleases or Holliday junction resolvases (HJRs). Genes for RecT/Redβ superfamily proteins are associated with genes encoding a λ-type exonuclease (LE) of the type II restric- tion enzyme fold, RecE, which also might be a divergent member of this fold, and a nuclease of the Endonuclease VII (EndoVII) fold [7,34] (Fig. 2). The ERF superfamily genes are associated with a RusA superfamily nuclease/ HJR and EndoVII fold nucleases (Fig. 4) [34]. Further- more, the Borrelia plasmids that encode ERF, also almost always additionally encode a λ-type exonuclease, even if it is not the adjacent gene. In a single instance, in the Gram- positive bacterium Ruminococcus albus, the gene encoding a RAD52 superfamily protein occurs adjacent to a gene for a λ-type exonuclease. These nucleases probably contribute to the repair process, in which SSAPs are involved, by pro- viding the initial break in the dsDNA and/or in digesting the nicked target to generate ssDNA. The RecT and Redβ family proteins often co-occur with the SbcC gene that encodes an ABC ATPase with a large coiled-coil segment. These proteins are known to cooper- ate with SbcD, nuclease of the calcineurin-like phosphoe- sterase superfamily and to degrade dsDNA in the 3' → 5' direction generating ssDNA [35,36]. It seems likely that RecT/Redβ proteins, at least in certain cases, function in conjunction with the SbcCD-pathway, by utilizing the sin- gle-stranded regions generated by the SbcCD nuclease. Additionally, several genes, whose functions are less clear, tend to co-occur with the genes coding for the SSAPs. Page 8 of 11 (page number not for citation purposes)