BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/8 Helix-1 Strand-1 Helix-2 Helix-3 Sec. Str. RAD52 family ---HHHHHHH-HH-----EEEE-----------------EEEE-HHHHHHHHHHHH----HHHHHHH-EEEEE-------------- Sec. Str. Bac family ---HHHHHHH-H-------EEEEE---------------EE---HHHHHHHHHHHH----HHHHEEE-------------------- RAD52_Kla_1172824 9 NGKDDIQTKL-DKKLGPEYISKRV---GFGS------SRVAYIEGWKAINLANQIFGYDGWSTEVKNVTIDFLDER----------- MUS11_NC_10039385 28 QEIATLQSRL-EKQLGPEYLSSRA---GPSG------QKVHYISSEKCIQLANEVFGFNGWSSSIQNIQVDFVDEHPQ--------- SPBC119.14_Sp_7491323 15 DSQQDKMTKLLAMQLGPEYISRRS---GPGG------GSVTYLEAWKAIELANEIFGFNGWSSSIQDIHVDYVEETK---------- RAD52_Sc_6323609 49 NHSEDIQTKL-DKKLGPEYISKRV---GFGT------SRIAYIEGWRVINLANQIFGYNGWSTEVKSVVIDFLDER----------- RAD22_Sp_1346948 24 EEFNFLQSSL-TRKLGPEYVSRRS---GPGG------FSVSYIESWKAIELANEIFGFNGWSSSIRSINVDFMDENKE--------- RAD52_Hs_14784422 34 EEYQAIQKAL-RQRLGPEYISSRM---AGGG------QKVCYIEGHRVINLANEMFGYNGWAHSITQQNVDFVDLN----------- RAD59_Sc_6320144 46 QRIGLLQSKI-ERYTYNIYHNNKY---GKHN------LSKLIP-GHALIQFANETFGYDGWRMDVIDVEARECQPFTAVNNGENTNT ORF252_BPul36_8248159 2 ADYEEQMLAL-QKPLQPDRVVWRVQQSGFSKQGKPWAMVLAYMDNRAVQERFDEVFGIAGWKNEFKTAPD----------------- CAC1936_Cab_15024916 1 DWGTYFKWQI-IRLLKASEIECRVQSVKKSG-----CVLLLYKDARCDMKILDETFGVLGWEREHQLVN------------------ DR0423_Dr_7472617 1 MKLSDVQKRL-QAPFPAHTVSWKPAAFNAERTR---ALLLAHVDARAVQDRLD-AVCPDDWSFEMEVVS------------------ Sparatyphi 3 VVLLMNLDQL-DEPFAAEDIEWRIQQSGKTGSGKVWAIVLAYVTNRAIMKRLDDVCGKAGWRNEYRDIPNN---------------- Ralbus 2 GRCYMNKLSI--RLLRADEIECRIGTISDKG-----LSLLLYKDARADMKILDETFGILGWRRTHQSID------------------ consensus/90% ......b..l..b.h.sp.lp.+....s...........l.a.ps..hbpbhsp.hG.sGW..php..................... HhH-1 HhH-2 Sec. Str. RAD52 family Sec. Str. Bac family RAD52_Kla_1172824 MUS11_NC_10039385 SPBC119.14_Sp_7491323 RAD52_Sc_6323609 RAD22_Sp_1346948 RAD52_Hs_14784422 RAD59_Sc_6320144 ORF252_BPul36_8248159 CAC1936_Cab_15024916 DR0423_Dr_7472617 Sparatyphi Ralbus consensus/90% Strand-2 Strand-3 Helix-4 Helix-5 Helix-6 Helix-7 ----EEEEEEEEEEEEE------EE---------------HHHHHHHH---HHHHHHHHHHHH-----HHHH-H-HHHHHHHHH------- --------EEEEEEEEE-----EEEE-----------------HH-HH-----HHHHHHHHHH-----HHHH---HHH------------- -QGRFSIGCTAIVRVSLAD---GTFREDIGYGTVENERRKASAFERAKKSAVTDALKRSLRGFGNALGNCLYDK-DFLAKIDKVKFDPPDF 160 -TLKINMGISVIMRVTLRD---GTYHEDLGYGHIENCKGKAAAFEKAKKEATTDALKRALRQFGNVLGNCIYDK-QYLAKVTKMKVEPTKF 181 -EKKFNVGISVIVRVTLKD---GSFHEDVGYGSIENCRVKALAYEKCKKEGTTDALKRALRNFGSSMGNCLYDK-RYIQKILKMAPAQAEF 168 -QGKFSIGCTAIVRVTLTS---GTYREDIGYGTVENERRKPAAFERAKKSAVTDALKRSLRGFGNALGNCLYDK-DFLAKIDKVKFDPPDF 200 -NGRISLGLSVIVRVTIKD---GAYHEDIGYGSIDNCRGKASAFEKCKKEGTTDALKRALRNFGNSLGNCMYDK-YYLREVGKMKPPTYHF 177 -NGKFYVGVCAFVRVQLKD---GSYHEDVGYGVSEGLKSKALSLEKARKEAVTDGLKRALRSFGNALGNCILDK-DYLRSLNKLPRQLPLE 185 SEVKYTVVAEAQVKVTLKD---GTNTQCGGLGRIT-LSSRGECYNRSKKEAVGDALKKALLSFE----KIILDY-ETKITNNYYVDGLYGS 203 ------GGTLCGISVKFGDE--WVTKWDGAENT---------QVE-AVKGGLSGSMKRAAVQWG--VGRYLYDLPTSFAQTSLEKTDGWNK 141 ------GNLFCTVRVYDEDKQMWISKQDVGTES---------NTE-KEKGQASDSFKRACFNFG--IGRELYTSPFIWIGLKPDEVTENKG 129 ------GAEVPTVKGRLTVL--GVTREDIGEAPEG-------SMA-AYKAAASDAMKRCAVQFG--IGRYLYDLPKQWADWDDARRGPKHL 137 ------GGVECGISIKIGSE--WVTKWDAAENT---------QVE-AVKGGRSGAMKRAAVQWG--IGRYLYNLEEGFAQISSDKKQGWHR 143 ------GNLYCTIELWDESKHEWIAKQDVGTES---------YAE-KEKGQASDSFKRACVSVG--IGRELYSAPFVWVSAQKCNIQRRSD 136 .......s..s.lplph.s....s.+bD.ubs...........hp.s.K.u.osuhKRsh..aG..hGphlhsb...h.p........... Figure 5 Multiple sequence alignment of the RAD52 protein superfamily. The coloring reflects the consensus at 90% conserva- tion. The coloring scheme and secondary structure assignment abbreviations are as in Fig. 1. Species abbreviations are as fol- lows: BPul36: Bacteriophage ul36. Cab: Clostridium acetobutylicum, Dr: Deinococcus radiodurans, Hs: Homo sapiens, Kla: Kluyveromyces lactis, NC: Neurospora crassa, Sc: Saccharomyces cerevisiae, Sp: Schizosaccharomyces pombe, Sparatyphi: Salmonella paratyphi A, Ralbus: Ruminococcus albus. The Shiga toxin-converting phage RAD52-like protein (gi: 17977996) is nearly identical to the Salmonella paratyphi A RAD52 like protein. The RAD52-like proteins from Bacteriophage ul36 (gi: 8248159) and Rumino- coccus albus are respectively adjacent to genes encoding the single-strand binding protein and the λ-type exonuclease. shows a quaternary organization similar to those of RecT/ Redβ and ERF [16,26]. However, RAD52-like proteins showed no detectable sequence similarity with either the ERF or the RecT/Redβ-like proteins. Sequence searches in- itiated with the conserved globular region of the eukaryo- tic RAD52 proteins readily detected their homologs from other eukaryotes and, at convergence, also retrieved from the database certain bacterial proteins, such as DR0423 from Deinococcus and CAC1936 from Clostridium respec- tively, with border-like statistical significance (e ~ .05). These bacterial proteins form a small family that is addi- tionally represented in Salmonella paratyphi A, the temper- ate bacteriophage u136 of Lactococcus lactis (ORF252- encoded protein) and a Shiga toxin-converting phage from E. coli. Iterative profile searches initiated with CAC1936 from Clostridium acetobutylicum and its S. para- typhi A ortholog correspondingly retrieved S. cerevisiae RAD52 and its eukaryotic homologs, with borderline e- values at convergence (~0.043). The alignment between these bacterial proteins and the eukaryotic Rad52 ho- mologs was co-linear throughout the entire length of their shared globular region and the Gibbs sampling procedure detected two motifs of greater than 20 residues, with a probability of chance occurrence in these proteins less than 10-18 (Fig. 5). In addition to the similar conservation pattern, separate secondary structure predictions for both the eukaryotic RAD52 family and their potential bacterial homologs showed a complete concordance of the predict- ed structural elements between RAD52 and the bacterial proteins, strongly suggesting that they all belong to a sin- gle homologous superfamily (hereinafter the RAD52 su- perfamily). Page 7 of 11 (page number not for citation purposes)