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/ Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. Eukaryotic Cell (1535-9778) 2005 imex curation Williams DR., McIntosh JR. Dewight.williams@vanderbilt.edu 12-MAR-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Williams DR., McIntosh JR. Dewight.williams@vanderbilt.edu Accepted 2007-MAR-28 by DAVET. 12-MAR-2007: Contacted by JYOTI. Eukaryotic Cell (1535-9778) 2005 Only protein-protein interactions imex curation Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. Eukaryotic Cell (1535-9778) 2005 imex curation Williams DR., McIntosh JR. Dewight.williams@vanderbilt.edu 12-MAR-2007: Contacted by JYOTI. Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast pull down pull down western blot western blot Immuno blot Immuno blot Williams DR., McIntosh JR. Looked for cell cycle dependence for this interaction. Cell lysates arrested by the mutations cdc25-22 (G2), cdc22- M45 (S phase), cdc21-M68 (S), or orp1-4 (pre-S) were used in this analysis (Fig. 2E). Dewight.williams@vanderbilt.edu Accepted 2007-MAR-28 by DAVET. 12-MAR-2007: Contacted by JYOTI. Eukaryotic Cell (1535-9778) 2005 Only protein-protein interactions imex curation Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. Eukaryotic Cell (1535-9778) 2005 imex curation Williams DR., McIntosh JR. Dewight.williams@vanderbilt.edu 12-MAR-2007: Contacted by JYOTI. Only protein-protein interactions schpm Schizosaccharomyces pombe (fission yeast) Fission yeast pull down pull down western blot western blot Immuno blot Immuno blot Williams DR., McIntosh JR. Expression of Mcl1-MHp was repressed by 10 mM thiamine (Fig. 2D, OFF). Because DNA might have mediated this weak interaction, the purified was digested material while it was still on the Talon beads with DNase I, followed by extensive washes (Fig. 2D). This treatment was not sufficient to disrupt the weak Mcl1-MHp interaction with the GFP-NT protein fragment. Dewight.williams@vanderbilt.edu Accepted 2007-MAR-28 by DAVET. 12-MAR-2007: Contacted by JYOTI. Eukaryotic Cell (1535-9778) 2005 Only protein-protein interactions imex curation Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. Eukaryotic Cell (1535-9778) 2005 imex curation Williams DR., McIntosh JR. Dewight.williams@vanderbilt.edu 12-MAR-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Williams DR., McIntosh JR. To determine whether the observed changes in mobility represented a loss of Pol1p from these Mcl1p complexes, a high-affinity antibody to GFP was used to precipitate Mcl1-GFP from the sucrose gradient fractions. Dewight.williams@vanderbilt.edu Accepted 2007-MAR-28 by DAVET. 12-MAR-2007: Contacted by JYOTI. Eukaryotic Cell (1535-9778) 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 1 5 unspecified role unspecified role bait bait n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role prey prey c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Figure 2C Mcl1p interacts with the N terminus of Pol1p both in vitro and in vivo. Two epitope-tagged Mcl1 proteins (diagrammed in Fig. 2A) were highly enriched in material recovered from yeast cellular lysates bound to an affinity matrix composed of an N-terminal Pol1 protein fragment fused to GST (GST-NT in Fig. 2B and C). This protein fragment contains amino acids 118 to 634 of Pol1p (47). None of these tagged alleles of Mcl1p interacted strongly with the C-terminal GST-Pol1p protein chimera (GST-CT in Fig. 2B and C) or GST (Fig. 2C). Such GST pulldown assays from whole-cell lysates demonstrate a high-affinity interaction between Mcl1p and the N terminus of Pol1p. There was strong interaction between Mcl1 and the n-terminus of Pol1. pol1-mcl1-1 1 5 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 6 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 physical association physical association Figure 2C Mcl1p interacts with the N terminus of Pol1p both in vitro and in vivo. Two epitope-tagged Mcl1 proteins (diagrammed in Fig. 2A) were highly enriched in material recovered from yeast cellular lysates bound to an affinity matrix composed of an N-terminal Pol1 protein fragment fused to GST (GST-NT in Fig. 2B and C). This protein fragment contains amino acids 118 to 634 of Pol1p (47). None of these tagged alleles of Mcl1p interacted strongly with the C-terminal GST-Pol1p protein chimera (GST-CT in Fig. 2B and C) or GST (Fig. 2C). Such GST pulldown assays from whole-cell lysates demonstrate a high-affinity interaction between Mcl1p and the N terminus of Pol1p. There was strong interaction between Mcl1 and the n-terminus of Pol1. pol1-mcl1-2 3 6 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 5 unspecified role unspecified role bait bait n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) physical association physical association Figure2D An in vivo interaction between the N terminus of Pol1p and Mcl1-MHp was tested by expression of the first 180 amino acids of Pol1p C-terminally fused to GFP (Fig. 2B, bottom) and expressed under the regulation of its endogenous promoter from a plasmid (pTB19) in nmt41mcl1-MH cells. Purification of Mcl1-MHp with Talon resin recovered only a fraction of the GFP-NT chimera (Fig. 2B) from cell lysates when cells were grown in the absence of 10 mM thiamine (Fig. 2D, ON) but did not recover any detectable GFP-NT when the expression of Mcl1-MHp was repressed by 10 mM thiamine (Fig. 2D, OFF). Because DNA might have mediated this weak interaction, we digested the purified material while it was still on the Talon beads with DNase I, followed by extensive washes (Fig. 2D). This treatment was not sufficient to disrupt the weak Mcl1-MHp interaction with the GFP-NT protein fragment. Expression of GFP with Mcl1-MHp does not recover GFP with Mcl1-MHp, demonstrating that the Pol1p fragment interaction was not induced by nonspecific interaction between GFP and Mcl1-MHp. There was weak in-vivo interaction between Mcl1 and the small n-terminus fragment of Pol1. pol1-mcl1-3 2 5 unspecified role unspecified role bait bait c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 6 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 physical association physical association Figure 2E Cell lysates arrested by the mutations cdc25-22 (G2), cdc22- M45 (S phase), cdc21-M68 (S), or orp1-4 (pre-S) were used in this analysis (Fig. 2E). In G2 and pre-S-phase arrests (cdc25-22 and orp1-4, respectively), the majority of the GFP-NT remained unbound to Mcl1-MHp recovered by cobalt-immobilized resin, but during S phase nearly all of the GFP-NT in the cellular lysate was recovered with the Mcl1-MHp. There was in-vivo interaction between Mcl1 and the small n-terminus fragment of Pol1 at S-phase of the cell cycle. pol1-mcl1-4 4 5 unspecified role unspecified role prey prey schpm Schizosaccharomyces pombe (fission yeast) Fission yeast 6 unspecified role unspecified role bait bait c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Figure 6B Immunoprecipitation of Mcl1-GFP from fraction 9 coprecipitates Pol1p from all genetic backgrounds except nmt1GST-cds1, which contained no Mcl1-GFP in fraction 9 (Fig. 6B, top panels). Pol1p also precipitated with Mcl1-GFP from the trailing fractions of rad3 and cds1 , indicating that the mobility differences observed in these mutants were not due to a loss of Pol1p interaction. In contrast, the slower-sedimenting material in hsk1-1312 and nmt1GST-cds1 fractions contained no detectable Pol1p (Fig. 6B, bottom panels), suggesting that in these backgrounds the association between Pol1p and Mcl1-GFP is weaker than in the other conditions. Analysis of the N-terminally-tagged Mcl1-MHp failed to detect any direct interaction with endogenous Pol1p, but immunoprecipitation of the C-terminally-tagged Mcl1-GFP does recover endogenous Pol1p since there was weak interaction detected between Mcl1-GFP and endogenous Pol1. pol1-mcl1-5 3 5 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role prey prey c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag anti tag western anti tag western blot anti tag western c-term range C-terminal range c-term range c-term range C-terminal range c-term range schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association Figure 6C To test this result in a different way, we used the GST-Pol1NT118�634 bound to GSH-Sepharose to collect Mcl1-GFP from 3 mg of total protein in whole-cell extracts from the above arrested strains. This technique also showed that Mcl1-GFP interacts with Pol1p in the mcl1-GFP, cds1 , and hsk1-1312 backgrounds. It was not, however, present when GST-Cds1p was overexpressed. GST-Pol1(118-634)-GSH-Sepharose coprecipitates Mcl1-GFP from mcl1-GFP, hsk1-1312 and cds1D but not from nmtGST-cds1 whole cell extracts.