IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe. Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe. Genetics (0016-6731) 2001 imex curation Tsutsui Y., Khasanov FK., Shinagawa H., Iwasaki H., Bashkirov VI. Figure 1A, Table 1, Table 2, Table 3, Figure 4. vibashikirov@mail.ru 14-Nov-2008: Contacted by JYOTI. orchard Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Tsutsui Y., Khasanov FK., Shinagawa H., Iwasaki H., Bashkirov VI. Figure 1A, Table 1, Table 2, Table 3, Figure 4. vibashikirov@mail.ru Accepted 2008-NOV-15 by ORCHARD. 14-Nov-2008: Contacted by JYOTI. Genetics (0016-6731) 2001 orchard Only protein-protein interactions imex curation Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe. Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe. Genetics (0016-6731) 2001 imex curation Tsutsui Y., Khasanov FK., Shinagawa H., Iwasaki H., Bashkirov VI. Figure 1A, Table 1, Table 2, Table 3, Figure 4. vibashikirov@mail.ru 14-Nov-2008: Contacted by JYOTI. orchard Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Tsutsui Y., Khasanov FK., Shinagawa H., Iwasaki H., Bashkirov VI. Figure 1A, Table 1, Table 2, Table 3, Figure 4. vibashikirov@mail.ru Accepted 2008-NOV-15 by ORCHARD. 14-Nov-2008: Contacted by JYOTI. Genetics (0016-6731) 2001 orchard Only protein-protein interactions imex curation Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe. Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe. Genetics (0016-6731) 2001 imex curation Tsutsui Y., Khasanov FK., Shinagawa H., Iwasaki H., Bashkirov VI. Figure 1A, Table 1, Table 2, Table 3, Figure 4. vibashikirov@mail.ru 14-Nov-2008: Contacted by JYOTI. orchard Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Tsutsui Y., Khasanov FK., Shinagawa H., Iwasaki H., Bashkirov VI. Figure 1A, Table 1, Table 2, Table 3, Figure 4. vibashikirov@mail.ru Accepted 2008-NOV-15 by ORCHARD. Genetics (0016-6731) 2001 orchard Only protein-protein interactions imex curation rad22_schpo DNA repair and recombination protein rad22 rad22 SPAC30D11.10 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MSFEQKQHVASEDQGHFNTAYSHEEFNFLQSSLTRKLGPEYVSRRSGPGGFSVSYIESWKAIELANEIFGFNGWSSSIRSINVDFMDENKENGRISLGLSVIVRVTIKDGAYHEDIGYGSIDNCRGKASAFEKCKKEGTTDALKRALRNFGNSLGNCMYDKYYLREVGKMKPPTYHFDSGDLFRKTDPAARESFIKKQKTLNSTRTVNNQPLVNKGEQLAPRRAAELNDEQTREIEMYADEELDNIFVEDDIIAHLAVAEDTAHPAANNHHSEKAGTQINNKDKGSHNSAKPVQRSHTYPVAVPQNTSDSVGNAVTDTSPKTLFDPLKPNTGTPSPKFISARAAAAAEGVVSAPFTNNFNPRLDSPSIRKTSIIDHSKSLPVQRASVLPIIKQSSQTSPVSNNSMIRDSESIINERKENIGLIGVKRSLHDSTTSHNKSDLMRTNSDPQSAMRSRENYDATVDKKAKKG E6B8B538D28B899D rti1_schpo DNA repair and recombination protein rti1 rti1 SPBC119.14 Rad twenty-two isogene 1 Rad22 homolog protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MGSLPDQSSCEEFTDSQQDKMTKLLAMQLGPEYISRRSGPGGGSVTYLEAWKAIELANEIFGFNGWSSSIQDIHVDYVEETKEKKFNVGISVIVRVTLKDGSFHEDVGYGSIENCRVKALAYEKCKKEGTTDALKRALRNFGSSMGNCLYDKRYIQKILKMAPAQAEFNYDNLLRANKRPYARFAQKVSTPIESHANKSVKLEHKNSIEKKISNVDKPISDLIENDIHESLPALQNPPIQSHSETDLYADEELDSILMHHERPPIPESPRVEEFEELLNQFEGDEKVSVDKIDAHDKMTEAQVVKIPPVQFMNARVAAAENPHIKHEGMAFQLHKKSNSILKSSNIDHNRSMPIRRPSLTSNNSANTFSTK 62280F122A2991B0 rad51_schpo DNA repair protein rhp51 rhp51 rad51 SPAC644.14c RAD51 homolog protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MADTEVEMQVSAADTNNNENGQAQSNYEYDVNVQDEEDEAAAGPMPLQMLEGNGITASDIKKIHEAGYYTVESIAYTPKRQLLLIKGISEAKADKLLGEASKLVPMGFTTATEYHIRRSELITITTGSKQLDTLLQGGVETGSITELFGEFRTGKSQICHTLAVTCQLPIDMGGGEGKCLYIDTEGTFRPVRLLAVADRYGLNGEEVLDNVAYARAYNADHQLELLQQAANMMSESRFSLLVVDSCTALYRTDFSGRGELSARQMHLARFMRTLQRLADEFGIAVVITNQVVAQVDGISFNPDPKKPIGGNILAHSSTTRLSLRKGRGEQRICKIYDSPCLPESEAIFAINSDGVGDPKEIIAPV 9F26EB9FA4F3C2BA 9F26EB9FA4F3C2BA rad54_schpo DNA repair protein rhp54 rhp54 rad54 SPAC15A10.03c RAD54 homolog 1 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MIQQPTTAKPRISTSSKLNTVLSKNKENVPGKLFKKFKCPSLVISEKRKELPLRKKPRVNYSEYGSVDGKYDSAYVSENVSGLATIKEANRLILNHERRDPSTVIKKQFSVPKPIKGHEDISKLCAHRPPPTLGMKRKVDFIPRPLYDPADEFAIVLYDPTTDADEIIPDIKEVLAEKRKKDELLKNRKGKKEISDSEPESDHDSCVSTDTVASCSTEQSLITSNTSKHRRPNKSLKDLLGIQKEKPPPPPVAVVIDPKLARILRPHQIEGVKFLYKCVTGRIDRCANGCIMADEMGLGKTLQCIALLWTLLKQSPQAGKPTIEKAIITCPSSLVKNWANELVKWLGKDAITPFILDGKSSKQELIMALQQWASVHGRQVTRPVLIASYETLRSYVEHLNNAEIGMLLCDEGHRLKNSDSLTFTALDKLNVQRRVILSGTPIQNDLSEYFSLLNFANPGLLGSRQEFRKNYEIPILKGRDADGTEKDKENGDAKLAELAKIVNRFIIRRTNDILSKYLPVKYEHVVFCNLSEFQLSLYKHFITSPEINKILRGTGSQPLKAIGLLKKICNHPDLLNLTEDLEGCEALFPPGFIPRELRGRDRNIDSSLSGKMLVLERMLYQIKQETDDKIVLISNYTSTLDLFEQLCRARGYKALRLDGTMNVNKRQRLVDTFNDPEKDAFVFLLSSKAGGCGINLIGANRLILFDPDWNPAADQQALARVWRDGQKKDCFVYRFIATGTIEEKIFQRQSHKQSLSSCVVDEAQDVERHFSLDNLRQLFQLNDHTVCETHETYKCKRCRDGKQFIRAPAMLYGDTSTWNHFTNPTLDRIEDHLLKREAGKQQVSTVFQYKSH 9DB3A247A57B0880 rad57_schpo DNA repair protein rhp57 rhp57 RAD57 homolog SPAC20H4.07 SPAC145.01 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MDISNYVDNFYFDEKIASAFELGEVSTVDLLTLDITELERRTHCSQSELLQLIEQISLLLQPVRCSASKVTSKYLTTGDVKLDETLHGGIPVGQLTEICGESGSGKSQFCMQLCLMVQLPLSLGGMNKAAVFISTESGLETKRLFELARYLPERYPKADKKDIIIKNPGDRVYTILCPDLESQEHIIQYQLPILFNRDKIGLVILDSVASNYRAELRYNRSKSHFRDLDNIAKRGNQLGKLAMTLRTLAHQHEAAVVIANQVSDRIPRDYDAIGLFSLDYQSQWFSGWDDTDPNPKIPSLGLVWTNNISTRLALIKKTDSATNNSGRIFRVVYSPNSPRLDVRICIGSVGIYSC 40131E247A087DB0 rad55_schpo DNA repair protein rhp55 rhp55 RAD55 homolog SPAC3C7.03c protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MLSSQHRLVTQPAIRAYEAFSAPGFGFNSKLLDDAFGGSGLKRGYISEVCGAPGMGKTSLALQITANALLSGSRVIWVETCQPIPMERLRQLLDNHVPSSQDEEEKCDTDELLNLLDVVYAPNLVNILAFLRNFDQEKHLKEIGLLIIDNLSMPIQLAYPTSPEDYAYLRLRRNTSKKSSLSDSSQKENTLTLNKENEFSSKDDSNFAFHNSSTKTTINRRKKAIGTISSLLSKITSSCYVAIFVTTQMTSKVVSGIGAKLIPLLSTNWLDNLSYRLILYSRHSTEESKDGQSRPSHQLLRYAFMAKQPPAHSAESELAFQLTSTGIQDYQSIPTNSSQRRKRSILECES 53DC65C0EC3836E1 rfa1_schpo Replication factor A protein 1 ssb1 rad11 rpa1 SPBC660.13c Single-stranded DNA-binding protein p68 subunit protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MAERLSVGALRIINTSDASSFPPNPILQVLTVKELNSNPTSGAPKRYRVVLSDSINYAQSMLSTQLNHLVAENKLQKGAFVQLTQFTVNVMKERKILIVLGLNVLTELGVMDKIGNPAGLETVDALRQQQNEQNNASAPRTGISTSTNSFYGNNAAATAPAPPPMMKKPAAPNSLSTIIYPIEGLSPYQNKWTIRARVTNKSEVKHWHNQRGEGKLFSVNLLDESGEIRATGFNDQVDAFYDILQEGSVYYISRCRVNIAKKQYTNVQNEYELMFERDTEIRKAEDQTAVPVAKFSFVSLQEVGDVAKDAVIDVIGVLQNVGPVQQITSRATSRGFDKRDITIVDQTGYEMRVTLWGKTAIEFSVSEESILAFKGVKVNDFQGRSLSMLTSSTMSVDPDIQESHLLKGWYDGQGRGQEFAKHSVISSTLSTTGRSAERKNIAEVQAEHLGMSETPDYFSLKGTIVYIRKKNVSYPACPAADCNKKVFDQGGSWRCEKCNKEYDAPQYRYIITIAVGDHTGQLWLNVFDDVGKLIMHKTADELNDLQENDENAFMNCMAEACYMPYIFQCRAKQDNFKGEMRVRYTVMSINQMDWKEESKRLINFIESAQ 5F2E77A0E12022EA 5F2E77A0E12022EA rad55-rad57 3 9 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association Figure 1A. Figure 1A shows that the AD-Rhp55 construct activated transcription of the lacZ reporter 24-fold when present in the cell together with DBD-Rhp57. Reciprocally, an even higher increase in beta-galactosidase activity (147-fold) was observed when the AD-Rhp57 fusion was combined with the DBD-Rhp55 construct. rad55-rad57-1 3 9 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association Figure 1A. Figure 1A shows that the AD-Rhp55 construct activated transcription of the lacZ reporter 24-fold when present in the cell together with DBD-Rhp57. Reciprocally, an even higher increase in beta-galactosidase activity (147-fold) was observed when the AD-Rhp57 fusion was combined with the DBD-Rhp55 construct. rad55-rad57-2 2 9 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag western blot western blot Immuno blot Immuno blot undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role bait bait physical association physical association Figure 1B. We used anti-Rhp57p antibodies for immunoprecipitation and showed that Rhp55 and Rhp57 proteins can be coprecipitated (Figure 1B, lanes 6). rad55-rad57-3 1 9 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag western blot western blot Immuno blot Immuno blot undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role prey prey physical association physical association Figure 1C. Rhp55p:Rhp57 complexes were also identified in reciprocal immunoprecipitation experiments using anti-HA antibodies to precipitate Rhp55 protein (Figure 1C, lanes 6). rad51-rad54-1 3 7 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey physical association physical association Table 1 and Table 2 The N-terminal fusions of the full-length Rad22, Rhp51, Rhp54, Rhp55, and Rhp57 proteins with DBD (bait) and AD (prey) of yeast two-hybrid vectors were constructed and pairwise interactions were tested. As shown in Table 1, rad51-rad51-9 3 6 unspecified role unspecified role bait bait c-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role prey prey n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 32 30 physical association physical association Table 2. Therefore, we extended our analysis of interactions involving Rhp51p by using truncated versions of the protein. The N-terminal domain of Rhp51p (aa 1-117) was fused with DBD and AD to generate Rhp51deltaC bait and prey plasmids (Figure 4). Likewise, the core and C-terminal domains of Rhp51p (114�365) were used to construct Rhp51deltaN bait and prey. The results of two-hybrid analysis with these constructs are shown in Table 2. Both Rhp51deltaN and Rhp51deltaC were found to interact strongly with the full-length Rhp51p (763-fold and 2300-fold increases, respectively), which is consistent with the ability of RecA/Rad51-family members to polymerize on DNA via monomer-monomer interaction. The c-terminus of rad51 (aa 114-365) interacts with full-length rad51 but even stronger with the n-terminus of rad51 (aa 1-117). rad51-rad51-10 3 6 unspecified role unspecified role bait bait n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role prey prey c-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 35 37 physical association physical association Table 2. Therefore, we extended our analysis of interactions involving Rhp51p by using truncated versions of the protein. The N-terminal domain of Rhp51p (aa 1-117) was fused with DBD and AD to generate Rhp51deltaC bait and prey plasmids (Figure 4). Likewise, the core and C-terminal domains of Rhp51p (114�365) were used to construct Rhp51deltaN bait and prey. The results of two-hybrid analysis with these constructs are shown in Table 2. Both Rhp51deltaN and Rhp51deltaC were found to interact strongly with the full-length Rhp51p (763-fold and 2300-fold increases, respectively), which is consistent with the ability of RecA/Rad51-family members to polymerize on DNA via monomer-monomer interaction. The n-terminus of rad51 (aa 1-117) interacts strongly with both the full-length rad51 and the c-terminus of rad51 (aa 114-365). rad51-rad57-1 3 8 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association Table 2. Therefore, we extended our analysis of interactions involving Rhp51p by using truncated versions of the protein. The N-terminal domain of Rhp51p (aa 1-117) was fused with DBD and AD to generate Rhp51deltaC bait and prey plasmids (Figure 4). Likewise, the core and C-terminal domains of Rhp51p (114�365) were used to construct Rhp51deltaN bait and prey. The results of two-hybrid analysis with these constructs are shown in Table 2. Both Rhp51deltaN and Rhp51deltaC were found to interact strongly with the full-length Rhp51p (763-fold and 2300-fold increases, respectively), which is consistent with the ability of RecA/Rad51-family members to polymerize on DNA via monomer-monomer interaction. The n-terminus of rad51 (aa 1-117) is able to interact (weakly) with full-length rad57. rad51-rad57-2 3 6 unspecified role unspecified role prey prey n-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role bait bait physical association physical association Table 2. Therefore, we extended our analysis of interactions involving Rhp51p by using truncated versions of the protein. The N-terminal domain of Rhp51p (aa 1-117) was fused with DBD and AD to generate Rhp51deltaC bait and prey plasmids (Figure 4). Likewise, the core and C-terminal domains of Rhp51p (114�365) were used to construct Rhp51deltaN bait and prey. The results of two-hybrid analysis with these constructs are shown in Table 2. Both Rhp51deltaN and Rhp51deltaC were found to interact strongly with the full-length Rhp51p (763-fold and 2300-fold increases, respectively), which is consistent with the ability of RecA/Rad51-family members to polymerize on DNA via monomer-monomer interaction. Rad57 was able to bind to the n-terminus of rad51(aa 1-117) but not the c-terminus of rad51 (aa 114-365) rad51-rad22-1 3 4 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association Table 1, Table 2. Therefore, we extended our analysis of interactions involving Rhp51p by using truncated versions of the protein. The N-terminal domain of Rhp51p (aa 1-117) was fused with DBD and AD to generate Rhp51deltaC bait and prey plasmids (Figure 4). Likewise, the core and C-terminal domains of Rhp51p (114�365) were used to construct Rhp51deltaN bait and prey. The results of two-hybrid analysis with these constructs are shown in Table 2. Both Rhp51deltaN and Rhp51deltaC were found to interact strongly with the full-length Rhp51p (763-fold and 2300-fold increases, respectively), which is consistent with the ability of RecA/Rad51-family members to polymerize on DNA via monomer-monomer interaction. Rad22 was able to interact more strongly with the c-terminus of rad51(aa 114-365) than the full-length rad51. rad51-rti1 3 5 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association Table 3. Indeed, strong interaction between Rti1p and Rhp51deltaNp was detected with both bait and prey Rti1p constructs (49-fold and 539-fold, respectively), which implicates the region of Rhp51 protein between amino acids 114 and 365 in binding with Rti1p (Table 3). rad51-rti1-1 3 5 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait c-terminus required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association Table 3. Indeed, strong interaction between Rti1p and Rhp51deltaNp was detected with both bait and prey Rti1p constructs (49-fold and 539-fold, respectively), which implicates the region of Rhp51 protein between amino acids 114 and 365 in binding with Rti1p (Table 3). rti1-rti1-2 3 5 unspecified role unspecified role prey prey 5 unspecified role unspecified role bait bait physical association physical association Table 3. Moreover, moderate but significant self-interactions of Rad22p-Rad22p (38-fold increase) and Rti1p-Rti1p (10-fold increase) were observed, suggesting that in vivo these proteins may exist as oligomers. There seems to be a weak self-interaction with rti1. rad11-rti1-3 3 10 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey physical association physical association Table 3. Finally, we found that Rad11p (Rpa1p) bait could interact strongly with Rti1p (73-fold increase) and less strongly with Rad22p (12-fold increase). Rad11 interacts strongly with Rti1. rad11-rad22 3 4 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait physical association physical association Table 3. Finally, we found that Rad11p (Rpa1p) bait could interact strongly with Rti1p (73-fold increase) and less strongly with Rad22p (12-fold increase). Rad11 interacts weakly with Rad22. rti1-rad22 3 4 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey physical association physical association Table 3. When Rad22p was used as the DBD and Rti1p as the AD fusion, their interaction was manifested by a 111-fold increase in beta-galactosidase activity compared to the control (Table 3). Rad22 interacts strongly with Rti1. rad22-rad22 3 4 unspecified role unspecified role bait bait 4 unspecified role unspecified role prey prey physical association physical association Table 3. Moreover, moderate but significant self-interactions of Rad22p-Rad22p (38-fold increase) and Rti1p-Rti1p (10-fold increase) were observed, suggesting that in vivo these proteins may exist as oligomers. Rad22 is able to interact with itself.