BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/8 ble-stranded DNAs followed by the exchange of DNA strands between the paired molecules. Experimental stud- ies have shown that members of the archetypal RecA fam- ily of recombinases are central to this reaction in all extant forms of life [1,2]. Studies in Escherichia coli have shown that, although RecA is the principle protein involved in pairing and strand ex- change, unrelated proteins, that have a much more restric- tive phyletic distribution, can also promote similar reactions in a RecA dependent or RecA-independent man- ner [3]. These alternative or additional mediators of ho- mologous recombination include the well-characterized prophage RecT, phage λ Redβ and phage P22 ERF proteins [4,5]. Similarly, in yeast and vertebrates, the RAD52 pro- tein is involved in the pairing and strand exchange reac- tion and can promote recombination in a RAD51 (the eukaryotic RecA homolog)-dependent or independent manner [6]. The RecT protein works in conjunction with the RecE-nuclease [7] and was initially described in genet- ic studies on the complemention of mutations in the RecBCD pathway of DNA repair [8–10]. Biochemically, RecT has been shown to bind single-stranded (ss) DNA 3' overhang regions generated by the RecE nuclease, and promote strand exchange between homologous DNA partners by assisting the pairing of complementary single- stranded regions [4,10]. The reaction catalyzed by the RecT/RecE system is similar to that described for the phage λ exonuclease (exo/Redβ) and the single-strand annealing protein Redβ. The similarity between these two systems is further extended by the observation that the RecT/E sys- tem can complement mutations in the λ exo/Redβ system [10,12]. In eukaryotes, RAD52 protein has been shown to exhibit properties similar to those of RecT and Redβ pro- teins: it binds ssDNA and promotes strand exchange via the pairing of complementary single strands [6,13]. In vit- ro studies on quaternary structures have shown that the single strand annealing proteins (SSAPs), RecT, Redβ, ERF and RAD52, form similar helical super-structures [14–17]. This has led to the proposal that RecT, Redβ, ERF and the eukaryotic RAD52 function in an analogous fashion, and even are "structural homologs" [14]. However, no sequence or secondary structural similarities have been noticed between different SSAPs and current understanding of their evolutionary history and phyletic range remains poor. Here, we describe the results of an in- depth sequence analysis of these proteins and delineate their evolutionary relationships and phyletic horizon in available genomes. We show that, in spite of the function- al similarities, and the similar quaternary structures, there are three distinct superfamilies of SSAPs, namely the RecT/ Redβ, RAD52 and ERF, that appear to be evolutionarily unrelated to each other. These superfamilies show a wide distribution in viral and cellular genomes, but appear to have originally evolved in large DNA bacteriophages. Through an analysis of the contextual information provid- ed by the predicted operons, in which the SSAPs occur, we predict several previously undetected functional connec- tions of these proteins, which might shed new light on the corresponding DNA repair/recombination pathways. Results RecT and Redβ are evolutionarily related and define a widespread family of DNA recombination proteins Several lines of evidence, including genetic analyses, and similarities in biochemistry and quaternary structures, suggest that the E. coli RecT and phage λ Redβ proteins are functionally equivalent as mediators of single-strand ex- change in DNA recombination [10,12]. However, no se- quence similarity has been detected between these proteins leaving their actual evolutionary relationships unresolved. In order to gain a better understanding of their functions and origins, we undertook a detailed se- quence analysis of these two proteins using iterative se- quence profile searches with the PSI-BLAST program with a inclusion threshold of .01 iterated until convergence. Such searches, with Redβ proteins from different lamb- doid bacteriophages as queries, retrieved not only other obvious Redβ homologs, but also the RecT protein family. For example, searches initiated with the Redβ homolog, PF161 protein (Genbank gi: 9836834 amino acids 1 to 188) from Borrelia hermsii[18], detected the E. coli RecT protein in the 5th iteration with significant expectation (e) values (3 × 10-3). Subsequent iterations retrieved several more RecT-related proteins from diverse sources. Further, transitive searches with the proteins detected in the above searches resulted in the identification of more divergent homologs, such as a protein termed the 'enterohemolysin associated protein' (EHAP1) from E. coli[19] and its or- thologs in Salmonella (Fig. 1). An examination of the pair- wise alignments generated by these searches showed that all these proteins shared a characteristic set of residues, in- cluding two highly conserved aromatic residues at the N- and C-termini, respectively, and two consecutive acidic residues near the C-terminus. These observations strongly suggested that RecT and Redβ, along with several other proteins, could be unified into a single protein superfami- ly with a core conserved domain of approximately 200 amino acid residues. A multiple alignment of all members of the RecT/Redβ su- perfamily was generated using the T_coffee program fol- lowed by adjustments based on the PSI-BLAST search results. This alignment was used to predict their secondary structure using the JPRED and PHD methods; these pre- dictions pointed to an α + β domain with a core of five β- strands and five α-helices (Fig. 1). Some of the strongest conservation is concentrated in the long helices, and the pattern includes some charged or polar residues, suggest- Page 2 of 11 (page number not for citation purposes)