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/ Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase alpha: evidence for a multifunctional role of Mcl1 in DNA replication and repair. Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase alpha: evidence for a multifunctional role of Mcl1 in DNA replication and repair. Curr. Genet. (0172-8083) 2005 imex curation Tsutsui Y., Morishita T., Natsume T., Yamashita K., Iwasaki H., Yamao F., Shinagawa H. In Y2H analysis Mcl1pDN (420�815 aa) and PolaDC (amino acids 1�867) were expressed as fusion proteins and the two independent transformants carrying DBD and AD fusion plasmids were assayed for b-galactosidase activity. ytsutsui@lab.nig.ac.jp 12-MAR-2007: Contacted by JYOTI. Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast anti tag coip anti tag coimmunoprecipitation anti tag coip predetermined predetermined participant predetermined Tsutsui Y., Morishita T., Natsume T., Yamashita K., Iwasaki H., Yamao F., Shinagawa H. ytsutsui@lab.nig.ac.jp Accepted 2007-MAR-28 by DAVET. 12-MAR-2007: Contacted by JYOTI. Curr. Genet. (0172-8083) 2005 Only protein-protein interactions imex curation Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase alpha: evidence for a multifunctional role of Mcl1 in DNA replication and repair. Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase alpha: evidence for a multifunctional role of Mcl1 in DNA replication and repair. Curr. Genet. (0172-8083) 2005 imex curation Tsutsui Y., Morishita T., Natsume T., Yamashita K., Iwasaki H., Yamao F., Shinagawa H. In Y2H analysis Mcl1pDN (420�815 aa) and PolaDC (amino acids 1�867) were expressed as fusion proteins and the two independent transformants carrying DBD and AD fusion plasmids were assayed for b-galactosidase activity. ytsutsui@lab.nig.ac.jp 12-MAR-2007: Contacted by JYOTI. 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., Morishita T., Natsume T., Yamashita K., Iwasaki H., Yamao F., Shinagawa H. In Y2H analysis Mcl1pDN (420�815 aa) and PolaDC (amino acids 1�867) were expressed as fusion proteins and the two independent transformants carrying DBD and AD fusion plasmids were assayed for b-galactosidase activity. ytsutsui@lab.nig.ac.jp Accepted 2007-MAR-28 by DAVET. 12-MAR-2007: Contacted by JYOTI. Curr. Genet. (0172-8083) 2005 Only protein-protein interactions imex curation dpoa_schpo DNA polymerase alpha catalytic subunit swi7 SPAC3H5.06c pol1 DNA polymerase I protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MRKRNAGIKPSQRFAELRALRQAGKTRASTYESKENEELYDNVSEEEYRKIVRQRLDEDDFVVDDNGAGYVDNGYDEWDQSHYSDEDDENEKGSSGRKRKKSKNVLAEPNQQIGAFFKANAIQPASRIANRKNNEKEDEFMAEILGSIDQDIPERLSTKKGNRSHTTSNAKRRSQKAASSISTTNVVSSDPKKYIPETVPSTPQPASSLPIPSSPPAALETEENVDSQPMELDEPELPVGSDAPIIDVEESIAMKPLMSHDQVDDTASLSKATISQQPLSPMTPLSSELDVSAFSEINSKIKNVDVPASSYSSPISKVSPSDVTEEDGSLFFFWMDYTEMYGSLCLFGKVYDKATKQYVSCFLKVDGIMRSLYFLPRPSSSSVSEDSIAAQTKDVYDEVANLLSKRGVKEWKSRVSKYKYAFELEDVPRTADYLEVIYSYSYPALPTDLTGSSFSHVFGTNTALFEQFVLSRRVMGPCWLKIQQPNFDAVKNASWCRVEIGCSSPQNISVSFEKNEITSKTPPMTVMSLAFRTLINKEQNKQEVVMISARIFENVDIEKGLPANDMPSYSFSLIRPLKQIFPNGFEKLARQHKSSIFCERSEVSLLNNFLNKVRTYDPDVYFGHDFEMCYSVLLSRLKERKIHNWSSIGRLRRSEWPRSFNRSSQQFVEKQIIAGRLMCDLSNDFGRSMIKAQSWSLSEIVLKELDIKRQDINQEKALQSWTDTAHGLLDYLVHCEIDTFFIAAVAFKIQMLQLSKNLTNIAGNSWARTLTGTRAERNEYILLHEFKKNGYIVPDKQQSIRRHAEAFGAEDGLQEESLGKKKDKYKGGLVFEPQKGLYETCILVMDFNSLYPSIIQEYNICFTTVDRSPSNSDSDDQIPDTPSASANQGIFPRLIANLVERRRQIKGLLKDNSATPTQRLQWDIQQQALKLTANSMYGCLGYTKSRFYARPLAVLITYKGREALMNTKELADQMGLQVIYGDTDSVMLNTNVTDKNHALRIGNEFKEKVNERYSKLEIDIDNVYQRMLLHAKKKYAALQLDSQGKPNLDVKGLDMKRREFCTLAKEASKFCLDQILSGELTETVIENIHSYLMDFSEKMRNGKFPANKFIIFNRLGKNPEDYPNGKTMPFVQVALKKKARGENVRVGDVIPFIIAGSDADGHPADRAYSPQEIMNTNSTLVIDYNYYLSHQILPPIERVIAPIEGTNRARLAECLGLDARKYYSHETSESSAFQRYESTLTDDQCFINVSPLLLKCPSCNATSFSLRSVKSLKETLYANTVECDCGYEYSDFTIILQFSSQLRDFINLYYEGILVCDDSSCGNRTRQMSVYGKRCCNKSCRGSVHFEYNDEQLYNQIKFLLKAVQTTTGATRNGIIRCNAINKNISRIMNKNAREFVDMGLIFSS 06082A3BAD6347C2 mcl1_schpo Minichromosome loss protein 1 mcl1 SPAPB1E7.02c slr3 DNA polymerase alpha accessory factor Mcl1 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MAGNRLVPRYAHTDGLTRLAYTRDGKFLLTVGSNQVIRKFQVGSDEEPDSIDNHQDPITGIAVAENYFCTCSEDATVCVYPIDSPTEHTLLARTTLPIRDVAYSVDGNWIAIASDETAVKVVSSTDSSQIFSLRPAKASNKHVTYSPNGNFLAVSSCNGILYFYDTQTRELIKFLTNTIASLEAESEICSKAAWHPKNGTFAVASTDHFVSVISPDDWLPLYKLLPKENHSGVTDISWSSNGMYIAASFKKGGILIWDTQSHEVVVELPYSTVVALAWQPFENVLSFTTNQGILYSCPDVIPKSILKEENDPTKPLTSSKSKNRTSKELDDLFGSDDEQSQNVNDLDGNSANEENEFINHDGLDSSLDLDGDSYMVDENDLNLAKKRKQKALIDRTTTIENGSSKRRLLQASIHKPVHTGSTPWQGNRRYLCLNLVGFIWTVQQDAEHNTITVEFHDETTHRKYHFVDDQKFEMACLDHEGALYASPATESSPGVIYYKAHVDWSRKSEWAMALPMENESPVTISLSSSVVLVCTSAGYVRVFSRQGFPISIHRSKHLPFVACSSFQDTIITIANDGLSSDGNSRLVYSIEDISRDEMLQTGDGVALPPQGTLESVFFSDVGDPYIYDSTGVLLVLMHWRIPGQAKWIPVLDTNELERRKSRQESYWPVTVADNQFHCILLKGASRYPYFPRPMFTEFDFRIPCNTNNPDASTSVPVLEELQLRNKLFLTLLEDSIGDGDVTEDEKISIARLEANIDKALLQLIQKACLEERIERVYELTKTLRRTTSIAAAQKIALHHSLTNVAEKIGNLLSNV D3AD3EF2D7997C54 pol1-mcl1-6 2 4 unspecified role unspecified role prey prey c-terminal required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 3 unspecified role unspecified role bait bait n-terminal 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 Figure 5B The possibility that Mcl1p interacts physically with Pola was examined using a yeast two-hybrid assay. The results shown in Fig. 5b indicate that Mcl1p interacts with Pola and the interaction is mediated by the C-terminal region of Mcl1p (amino acids 420�815) and the Nterminal region of Pola (amino acids 1�866), indicating that WD repeats in Mcl1p are not involved in this interaction. There was strong interaction mediated by the C-terminal of Mcl1 and the N-terminal of Pol1. pol1-mcl1-7 2 4 unspecified role unspecified role bait bait c-terminal required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 3 unspecified role unspecified role prey prey n-terminal 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 Figure 5B The possibility that Mcl1p interacts physically with Pola was examined using a yeast two-hybrid assay. The results shown in Fig. 5b indicate that Mcl1p interacts with Pola and the interaction is mediated by the C-terminal region of Mcl1p (amino acids 420�815) and the Nterminal region of Pola (amino acids 1�866), indicating that WD repeats in Mcl1p are not involved in this interaction. There was strong interaction mediated by the C-terminal of Mcl1 and the N-terminal of Pol1. pol1-mcl1-8 1 3 unspecified role unspecified role bait bait n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag anti tag western anti tag western blot anti tag western n-term range n-terminal range n-term range n-term range n-terminal range n-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 4 unspecified role unspecified role prey prey n-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag anti tag western anti tag western blot anti tag western n-term range n-terminal range n-term range n-term range n-terminal range n-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Figure 5C There was strong interaction between Mcl1 and Pol1. The physical interaction between Mcl1p and Pola was also examined in cell extracts using HA-tagged Mcl1p and 6xHis-Myc-tagged Pola. The eptitope-tagged proteins were co-expressed from two different plasmids in wild-type cells with no adverse effect on cell viability or growth. Crude cell extracts were prepared and immunoprecipitated with antibody to the HA or Myc epitope tag (Fig. 5c). Anti-HA antibody co-precipitated Mcl1p and Pola (Fig. 5c, lane 11) and similar results were observed with anti-Myc antibody (lane 12), suggesting that Mcl1p and Pola interact physically in vivo. However, only a small fraction of Mcl1pD in the extract co-precipitated with PolaD (and vice versa), suggesting that the complex between them may not be very stable or may be transient. pol1-mcl1-9 1 3 unspecified role unspecified role prey prey n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag anti tag western anti tag western blot anti tag western n-term range n-terminal range n-term range n-term range n-terminal range n-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 4 unspecified role unspecified role bait bait n-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag anti tag western anti tag western blot anti tag western n-term range n-terminal range n-term range n-term range n-terminal range n-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Figure 5C There was strong interaction between Mcl1 and Pol1. The physical interaction between Mcl1p and Pola was also examined in cell extracts using HA-tagged Mcl1p and 6xHis-Myc-tagged Pola. The eptitope-tagged proteins were co-expressed from two different plasmids in wild-type cells with no adverse effect on cell viability or growth. Crude cell extracts were prepared and immunoprecipitated with antibody to the HA or Myc epitope tag (Fig. 5c). Anti-HA antibody co-precipitated Mcl1p and Pola (Fig. 5c, lane 11) and similar results were observed with anti-Myc antibody (lane 12), suggesting that Mcl1p and Pola interact physically in vivo. However, only a small fraction of Mcl1pD in the extract co-precipitated with PolaD (and vice versa), suggesting that the complex between them may not be very stable or may be transient.