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/ http://www.ebi.ac.uk/ williams-2005a-1 Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Williams DR., McIntosh JR. Dewight.williams@vanderbilt.edu Eukaryotic Cell (1535-9778) 2005 Only protein-protein interactions imex curation williams-2005a-2 Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. 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 Eukaryotic Cell (1535-9778) 2005 Only protein-protein interactions imex curation williams-2005a-3 Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. 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 Eukaryotic Cell (1535-9778) 2005 Only protein-protein interactions imex curation williams-2005a-4 Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival. 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 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 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 pol1-mcl1 GST pull down showing interaction between pol1 and mcl1 in SchPombe. 594329 N/A 594333 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 false 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 false ecoli Escherichia coli (strain K12) N/A 594334 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 false schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association false false false Figure 2C There was strong interaction between Mcl1 and the n-terminus of Pol1. pol1-mcl1-1 GST pull down showing interaction between pol1 and mcl1 in SchPombe. 594329 N/A 594333 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 false 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 false ecoli Escherichia coli (strain K12) N/A 594334 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 false schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association false false false Figure 2C There was strong interaction between Mcl1 and the n-terminus of Pol1. pol1-mcl1-2 GST pull down showing interaction between pol1 and mcl1 in SchPombe. 594330 N/A 594334 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 false schpm Schizosaccharomyces pombe (fission yeast) Fission yeast N/A 594333 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 false 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 false ecoli Escherichia coli (strain K12) physical association physical association false false false Figure2D There was weak in-vivo interaction between Mcl1 and the small n-terminus fragment of Pol1. pol1-mcl1-5 GST pull down showing interaction between pol1 and mcl1 in SchPombe. 594330 N/A 594333 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 false 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 false ecoli Escherichia coli (strain K12) N/A 594334 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 false schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association false false false Figure 6C GST-Pol1(118-634)-GSH-Sepharose coprecipitates Mcl1-GFP from mcl1-GFP, hsk1-1312 and cds1D but not from nmtGST-cds1 whole cell extracts. pol1-mcl1-3 GST pull down showing interaction between pol1 and mcl1 during the cell cycle in SchPombe. 594331 N/A 594333 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 false 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 false ecoli Escherichia coli (strain K12) N/A 594334 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 false schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association false false false Figure 2E 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 anti-tag coip showing interaction between mcl1 and endogenous pol1 in SchPombe. 594332 N/A 594333 unspecified role unspecified role prey prey schpm Schizosaccharomyces pombe (fission yeast) Fission yeast N/A 594334 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 false schpm Schizosaccharomyces pombe (fission yeast) Fission yeast physical association physical association false false false Figure 6B 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.