MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation. Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation. EMBO J. (0261-4189) 2012 imex curation van Deursen F., Sengupta S., De Piccoli G., Sanchez-Diaz A., Labib K. Only protein-protein interactions yeast Saccharomyces cerevisiae 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 van Deursen F., Sengupta S., De Piccoli G., Sanchez-Diaz A., Labib K. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation. Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation. EMBO J. (0261-4189) 2012 imex curation van Deursen F., Sengupta S., De Piccoli G., Sanchez-Diaz A., Labib K. Only protein-protein interactions yeast Saccharomyces cerevisiae Baker's yeast anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot van Deursen F., Sengupta S., De Piccoli G., Sanchez-Diaz A., Labib K. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation mcm4_yeast DNA replication licensing factor MCM4 MCM4 HCD21 YPR019W YP9531.13 CDC54 Cell division control protein 54 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSQQSSSPTKEDNNSSSPVVPNPDSVPPQLSSPALFYSSSSSQGDIYGRNNSQNLSQGEGNIRAAIGSSPLNFPSSSQRQNSDVFQSQGRQGRIRSSASASGRSRYHSDLRSDRALPTSSSSLGRNGQNRVHMRRNDIHTSDLSSPRRIVDFDTRSGVNTLDTSSSSAPPSEASEPLRIIWGTNVSIQECTTNFRNFLMSFKYKFRKILDEREEFINNTTDEELYYIKQLNEMRELGTSNLNLDARNLLAYKQTEDLYHQLLNYPQEVISIMDQTIKDCMVSLIVDNNLDYDLDEIETKFYKVRPYNVGSCKGMRELNPNDIDKLINLKGLVLRSTPVIPDMKVAFFKCNVCDHTMAVEIDRGVIQEPARCERIDCNEPNSMSLIHNRCSFADKQVIKLQETPDFVPDGQTPHSISLCVYDELVDSCRAGDRIEVTGTFRSIPIRANSRQRVLKSLYKTYVDVVHVKKVSDKRLDVDTSTIEQELMQNKVDHNEVEEVRQITDQDLAKIREVAAREDLYSLLARSIAPSIYELEDVKKGILLQLFGGTNKTFTKGGRYRGDINILLCGDPSTSKSQILQYVHKITPRGVYTSGKGSSAVGLTAYITRDVDTKQLVLESGALVLSDGGVCCIDEFDKMSDSTRSVLHEVMEQQTISIAKAGIITTLNARSSILASANPIGSRYNPNLPVTENIDLPPPLLSRFDLVYLVLDKVDEKNDRELAKHLTNLYLEDKPEHISQDDVLPVEFLTMYISYAKEHIHPIITEAAKTELVRAYVGMRKMGDDSRSDEKRITATTRQLESMIRLAEAHAKMKLKNVVELEDVQEAVRLIRSAIKDYATDPKTGKIDMNLVQTGKSVIQRKLQEDLSREIMNVLKDQASDSMSFNELIKQINEHSQDRVESSDIQEALSRLQQEDKVIVLGEGVRRSVRLNNRV 382D542AE38975E3 382D542AE38975E3 mcm3_yeast DNA replication licensing factor MCM3 MCM3 YEL032W SYGP-ORF23 Minichromosome maintenance protein 3 protein protein yeast Saccharomyces cerevisiae Baker's yeast MEGSTGFDGDATTFFAPDAVFGDRVRRFQEFLDTFTSYRDSVRSIQVYNSNNAANYNDDQDDADERDLLGDDDGDDLEKEKKAASSTSLNILPHRIIISLDDLREFDRSFWSGILVEPAYFIPPAEKALTDLADSMDDVPHPNASAVSSRHPWKLSFKGSFGAHALSPRTLTAQHLNKLVSVEGIVTKTSLVRPKLIRSVHYAAKTGRFHYRDYTDATTTLTTRIPTPAIYPTEDTEGNKLTTEYGYSTFIDHQRITVQEMPEMAPAGQLPRSIDVILDDDLVDKTKPGDRVNVVGVFKSLGAGGMNQSNSNTLIGFKTLILGNTVYPLHARSTGVAARQMLTDFDIRNINKLSKKKDIFDILSQSLAPSIYGHDHIKKAILLMLMGGVEKNLENGSHLRGDINILMVGDPSTAKSQLLRFVLNTASLAIATTGRGSSGVGLTAAVTTDRETGERRLEAGAMVLADRGVVCIDEFDKMTDVDRVAIHEVMEQQTVTIAKAGIHTTLNARCSVIAAANPVFGQYDVNRDPHQNIALPDSLLSRFDLLFVVTDDINEIRDRSISEHVLRTHRYLPPGYLEGEPVRERLNLSLAVGEDADINPEEHSNSGAGVENEGEDDEDHVFEKFNPLLQAGAKLAKNKGNYNGTEIPKLVTIPFLRKYVQYAKERVIPQLTQEAINVIVKNYTDLRNDDNTKKSPITARTLETLIRLATAHAKVRLSKTVNKVDAKVAANLLRFALLGEDIGNDIDEEESEYEEALSKRSPQKSPKKRQRVRQPASNSGSPIKSTPRRSTASSVNATPSSARRILRFQDDEQNAGEDDNDIMSPLPADEEAELQRRLQLGLRVSPRRREHLHAPEEGSSGPLTEVGTPRLPNVSSAGQDDEQQQSVISFDNVEPGTISTGRLSLISGIIARLMQTEIFEEESYPVASLFERINEELPEEEKFSAQEYLAGLKIMSDRNNLMVADDKVWRV 43DD4DACAF4456DC 43DD4DACAF4456DC mcm6_yeast DNA replication licensing factor MCM6 YGL201C MCM6 Minichromosome maintenance protein 6 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSPFPADTPSSNRPSNSSPPPSSIGAGFGSSSGLDSQIGSRLHFPSSSQPHVSNSQTGPFVNDSTQFSSQRLQTDGSATNDMEGNEPARSFKSRALNHVKKVDDVTGEKVREAFEQFLEDFSVQSTDTGEVEKVYRAQIEFMKIYDLNTIYIDYQHLSMRENGALAMAISEQYYRFLPFLQKGLRRVVRKYAPELLNTSDSLKRSEGDEGQADEDEQQDDDMNGSSLPRDSGSSAAPGNGTSAMATRSITTSTSPEQTERVFQISFFNLPTVHRIRDIRSEKIGSLLSISGTVTRTSEVRPELYKASFTCDMCRAIVDNVEQSFKYTEPTFCPNPSCENRAFWTLNVTRSRFLDWQKVRIQENANEIPTGSMPRTLDVILRGDSVERAKPGDRCKFTGVEIVVPDVTQLGLPGVKPSSTLDTRGISKTTEGLNSGVTGLRSLGVRDLTYKISFLACHVISIGSNIGASSPDANSNNRETELQMAANLQANNVYQDNERDQEVFLNSLSSDEINELKEMVKDEHIYDKLVRSIAPAVFGHEAVKKGILLQMLGGVHKSTVEGIKLRGDINICVVGDPSTSKSQFLKYVVGFAPRSVYTSGKASSAAGLTAAVVRDEEGGDYTIEAGALMLADNGICCIDEFDKMDISDQVAIHEAMEQQTISIAKAGIHATLNARTSILAAANPVGGRYNRKLSLRGNLNMTAPIMSRFDLFFVILDDCNEKIDTELASHIVDLHMKRDEAIEPPFSAEQLRRYIKYARTFKPILTKEARSYLVEKYKELRKDDAQGFSRSSYRITVRQLESMIRLSEAIARANCVDEITPSFIAEAYDLLRQSIIRVDVDDVEMDEEFDNIESQSHAASGNNDDNDDGTGSGVITSEPPADIEEGQSEATARPGTSEKKKTTVTYDKYVSMMNMIVRKIAEVDREGAEELTAVDIVDWYLLQKENDLGSLAEYWEERRLAFKVIKRLVKDRILMEIHGTRHNLRDLENEENENNKTVYVIHPNCEVLDQLEPQDSS 03AB793134E64A50 03AB793134E64A50 mcm5_yeast Minichromosome maintenance protein 5 MCM5 CDC46 YLR274W L9328.1 Cell division control protein 46 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSFDRPEIYSAPVLQGESPNDDDNTEIIKSFKNFILEFRLDSQFIYRDQLRNNILVKNYSLTVNMEHLIGYNEDIYKKLSDEPSDIIPLFETAITQVAKRISILSRAQSANNNDKDPENTSMDTDSLLLNSLPTFQLILNSNANQIPLRDLDSEHVSKIVRLSGIIISTSVLSSRATYLSIMCRNCRHTTSITINNFNSITGNTVSLPRSCLSTIESESSMANESNIGDESTKKNCGPDPYIIIHESSKFIDQQFLKLQEIPELVPVGEMPRNLTMTCDRYLTNKVIPGTRVTIVGIYSIYNSKNGAGSGRSGGGNGGSGVAIRTPYIKILGIQSDVETSSIWNSVTMFTEEEEEEFLQLSRNPKLYEILTNSIAPSIFGNEDIKKAIVCLLMGGSKKILPDGMRLRGDINVLLLGDPGTAKSQLLKFVEKVSPIAVYTSGKGSSAAGLTASVQRDPMTREFYLEGGAMVLADGGVVCIDEFDKMRDEDRVAIHEAMEQQTISIAKAGITTVLNSRTSVLAAANPIYGRYDDLKSPGDNIDFQTTILSRFDMIFIVKDDHNEERDISIANHVINIHTGNANAMQNQQEENGSEISIEKMKRYITYCRLKCAPRLSPQAAEKLSSNFVTIRKQLLINELESTERSSIPITIRQLEAIIRITESLAKLELSPIAQERHVDEAIRLFQASTMDAASQDPIGGLNQASGTSLSEIRRFEQELKRRLPIGWSTSYQTLRREFVDTHRFSQLALDKALYALEKHETIQLRHQGQNIYRSGV 01D9DE208A091CF2 01D9DE208A091CF2 cdc45_yeast Cell division control protein 45 CDC45 YLR103C L8004.11 SLD4 protein protein yeast Saccharomyces cerevisiae Baker's yeast MYYGISQFSEAYNKILRNSSSHSSCQLVIFVSCLNIDALCATKMLSLLFKKQLVQSQIVPIFGYSELRRHYSQLDDNINSLLLVGFGGVIDLEAFLEIDPQEYVIDTDEKSGEQSFRRDIYVLDAHRPWNLDNIFGSQIIQCFDDGTVDDTLGEQKEAYYKLLELDEESGDDELSGDENDNNGGDDEATDADEVTDEDEEDEDETISNKRGNSSIGPNDLSKRKQRKKQIHEYEGVLEEYYSQGTTVVNSISAQIYSLLSAIGETNLSNLWLNILGTTSLDIAYAQVYNRLYPLLQDEVKRLTPSSRNSVKTPDTLTLNIQPDYYLFLLRHSSLYDSFYYSNYVNAKLSLWNENGKKRLHKMFARMGIPLSTAQETWLYMDHSIKRELGIIFDKNLDRYGLQDIIRDGFVRTLGYRGSISASEFVEALTALLEVGNSTDKDSVKINNDNNDDTDGEEEEDNSAQKLTNLRKRWVSNFWLSWDALDDRKVELLNRGIQLAQDLQRAIFNTGVAILEKKLIKHLRIYRLCVLQDGPDLDLYRNPLTLLRLGNWLIECCAESEDKQLLPMVLASIDENTDTYLVAGLTPRYPRGLDTIHTKKPILNNFSMAFQQITAETDAKVRIDNFESSIIEIRREDLSPFLEKLTLSGLL 6500DDD62EC4E8EE 6500DDD62EC4E8EE mcm7_yeast DNA replication licensing factor MCM7 MCM7 YBR202W YBR1441 CDC47 Cell division control protein 47 Minichromosome maintenance protein 7 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSAALPSIQLPVDYNNLFNEITDFLVTFKQDTLSSDATRNENEDENLDAENIEQHLLEKGPKYMAMLQKVANRELNSVIIDLDDILQYQNEKFLQGTQADDLVSAIQQNANHFTELFCRAIDNNMPLPTKEIDYKDDVLDVILNQRRLRNERMLSDRTNEIRSENLMDTTMDPPSSMNDALREVVEDETELFPPNLTRRYFLYFKPLSQNCARRYRKKAISSKPLSVRQIKGDFLGQLITVRGIITRVSDVKPAVEVIAYTCDQCGYEVFQEVNSRTFTPLSECTSEECSQNQTKGQLFMSTRASKFSAFQECKIQELSQQVPVGHIPRSLNIHVNGTLVRSLSPGDIVDVTGIFLPAPYTGFKALKAGLLTETYLEAQFVRQHKKKFASFSLTSDVEERVMELITSGDVYNRLAKSIAPEIYGNLDVKKALLLLLVGGVDKRVGDGMKIRGDINVCLMGDPGVAKSQLLKAICKISPRGVYTTGKGSSGVGLTAAVMKDPVTDEMILEGGALVLADNGICCIDEFDKMDESDRTAIHEVMEQQTISISKAGINTTLNARTSILAAANPLYGRYNPRLSPLDNINLPAALLSRFDILFLMLDIPSRDDDEKLAEHVTYVHMHNKQPDLDFTPVEPSKMREYIAYAKTKRPVMSEAVNDYVVQAYIRLRQDSKREMDSKFSFGQATPRTLLGIIRLSQALAKLRLADMVDIDDVEEALRLVRVSKESLYQETNKSKEDESPTTKIFTIIKKMLQETGKNTLSYENIVKTVRLRGFTMLQLSNCIQEYSYLNVWHLINEGNTLKFVDDGTMDTDQEDSLVSTPKLAPQTTASANVSAQDSDIDLQDA ADA66C719D96DB4A ADA66C719D96DB4A pob3_yeast FACT complex subunit POB3 YML069W Facilitates chromatin transcription complex subunit POB3 POB3 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSTDFDRIYLNQSKFSGRFRIADSGLGWKISTSGGSAANQARKPFLLPATELSTVQWSRGCRGYDLKINTKNQGVIQLDGFSQDDYNLIKNDFHRRFNIQVEQREHSLRGWNWGKTDLARNEMVFALNGKPTFEIPYARINNTNLTSKNEVGIEFNIQDEEYQPAGDELVEMRFYIPGVIQTNVDENMTKKEESSNEVVPKKEDGAEGEDVQMAVEEKSMAEAFYEELKEKADIGEVAGDAIVSFQDVFFTTPRGRYDIDIYKNSIRLRGKTYEYKLQHRQIQRIVSLPKADDIHHLLVLAIEPPLRQGQTTYPFLVLQFQKDEETEVQLNLEDEDYEENYKDKLKKQYDAKTHIVLSHVLKGLTDRRVIVPGEYKSKYDQCAVSCSFKANEGYLYPLDNAFFFLTKPTLYIPFSDVSMVNISRAGQTSTSSRTFDLEVVLRSNRGSTTFANISKEEQQLLEQFLKSKNLRVKNEDREVQERLQTALGSDSDEEDINMGSAGEDDESVDEDFQVSSDNDADEVAEEFDSDAALSDAEGGSDEERPSKKPKVE 02C04F620C7B1450 02C04F620C7B1450 dpoa_yeast DNA polymerase alpha catalytic subunit A POL1 CDC17 DNA polymerase alpha:primase complex p180 subunit DNA polymerase I subunit A N2181 YNL102W protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSKSEKLEKLRKLQAARNGTSIDDYEGDESDGDRIYDEIDEKEYRARKRQELLHDDFVVDDDGVGYVDRGVEEDWREVDNSSSDEDTGNLASKDSKRKKNIKREKDHQITDMLRTQHSKSTLLAHAKKSQKKSIPIDNFDDILGEFESGEVEKPNILLPSKLRENLNSSPTSEFKSSIKRVNGNDESSHDAGISKKVKIDPDSSTDKYLEIESSPLKLQSRKLRYANDVQDLLDDVENSPVVATKRQNVLQDTLLANPPSAQSLADEEDDEDSDEDIILKRRTMRSVTTTRRVNIDSRSNPSTSPFVTAPGTPIGIKGLTPSKSLQSNTDVATLAVNVKKEDVVDPETDTFQMFWLDYCEVNNTLILFGKVKLKDDNCVSAMVQINGLCRELFFLPREGKTPTDIHEEIIPLLMDKYGLDNIRAKPQKMKYSFELPDIPSESDYLKVLLPYQTPKSSRDTIPSDLSSDTFYHVFGGNSNIFESFVIQNRIMGPCWLDIKGADFNSIRNASHCAVEVSVDKPQNITPTTTKTMPNLRCLSLSIQTLMNPKENKQEIVSITLSAYRNISLDSPIPENIKPDDLCTLVRPPQSTSFPLGLAALAKQKLPGRVRLFNNEKAMLSCFCAMLKVEDPDVIIGHRLQNVYLDVLAHRMHDLNIPTFSSIGRRLRRTWPEKFGRGNSNMNHFFISDICSGRLICDIANEMGQSLTPKCQSWDLSEMYQVTCEKEHKPLDIDYQNPQYQNDVNSMTMALQENITNCMISAEVSYRIQLLTLTKQLTNLAGNAWAQTLGGTRAGRNEYILLHEFSRNGFIVPDKEGNRSRAQKQRQNEENADAPVNSKKAKYQGGLVFEPEKGLHKNYVLVMDFNSLYPSIIQEFNICFTTVDRNKEDIDELPSVPPSEVDQGVLPRLLANLVDRRREVKKVMKTETDPHKRVQCDIRQQALKLTANSMYGCLGYVNSRFYAKPLAMLVTNKGREILMNTRQLAESMNLLVVYGDTDSVMIDTGCDNYADAIKIGLGFKRLVNERYRLLEIDIDNVFKKLLLHAKKKYAALTVNLDKNGNGTTVLEVKGLDMKRREFCPLSRDVSIHVLNTILSDKDPEEALQEVYDYLEDIRIKVETNNIRIDKYKINMKLSKDPKAYPGGKNMPAVQVALRMRKAGRVVKAGSVITFVITKQDEIDNAADTPALSVAERAHALNEVMIKSNNLIPDPQYYLEKQIFAPVERLLERIDSFNVVRLSEALGLDSKKYFRREGGNNNGEDINNLQPLETTITDVERFKDTVTLELSCPSCDKRFPFGGIVSSNYYRVSYNGLQCKHCEQLFTPLQLTSQIEHSIRAHISLYYAGWLQCDDSTCGIVTRQVSVFGKRCLNDGCTGVMRYKYSDKQLYNQLLYFDSLFDCEKNKKQELKPIYLPDDLDYPKEQLTESSIKALTEQNRELMETGRSVVQKYLNDCGRRYVDMTSIFDFMLN 50C9032DBE95B5AE 50C9032DBE95B5AE csm3_yeast Chromosome segregation in meiosis protein 3 CSM3 YM9796.01 YMR048W protein protein yeast Saccharomyces cerevisiae Baker's yeast MDQDFDSLLLGFNDSDSVQKDPTVPNGLDGSVVDPTIADPTAITARKRRPQVKLTAEKLLSDKGLPYVLKNAHKRIRISSKKNSYDNLSNIIQFYQLWAHELFPKAKFKDFMKICQTVGKTDPVLREYRVSLFRDEMGMSFDVGTRETGQDLERQSPMVEEHVTSAEERPIVADSFAQDKRNVNNVDYDNDEDDDIYHLSYRNRRGRVLDERGNNETVLNNVVPPKEDLDALLKTFRVQGPVGLEENEKKLLLGWLDAHRKMEKGSMTEEDVQLIQSLEEWEMNDIEGQHTHYDLLPGGDEFGVDQDELDAMKEMGF 0B92F561F91197AE 0B92F561F91197AE mcm2_yeast DNA replication licensing factor MCM2 MCM2 YBL023C YBL0438 Minichromosome maintenance protein 2 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSDNRRRRREEDDSDSENELPPSSPQQHFRGGMNPVSSPIGSPDMINPEGDDNEVDDVPDIDEVEEQMNEVDLMDDNMYEDYAADHNRDRYDPDQVDDREQQELSLSERRRIDAQLNERDRLLRNVAYIDDEDEEQEGAAQLDEMGLPVQRRRRRRQYEDLENSDDDLLSDMDIDPLREELTLESLSNVKANSYSEWITQPNVSRTIARELKSFLLEYTDETGRSVYGARIRTLGEMNSESLEVNYRHLAESKAILALFLAKCPEEMLKIFDLVAMEATELHYPDYARIHSEIHVRISDFPTIYSLRELRESNLSSLVRVTGVVTRRTGVFPQLKYVKFNCLKCGSILGPFFQDSNEEIRISFCTNCKSKGPFRVNGEKTVYRNYQRVTLQEAPGTVPPGRLPRHREVILLADLVDVSKPGEEVEVTGIYKNNYDGNLNAKNGFPVFATIIEANSIKRREGNTANEGEEGLDVFSWTEEEEREFRKISRDRGIIDKIISSMAPSIYGHRDIKTAVACSLFGGVPKNVNGKHSIRGDINVLLLGDPGTAKSQILKYVEKTAHRAVFATGQGASAVGLTASVRKDPITKEWTLEGGALVLADKGVCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTTLQARCSIIAAANPNGGRYNSTLPLAQNVSLTEPILSRFDILCVVRDLVDEEADERLATFVVDSHVRSHPENDEDREGEELKNNGESAIEQGEDEINEQLNARQRRLQRQRKKEEEISPIPQELLMKYIHYARTKIYPKLHQMDMDKVSRVYADLRRESISTGSFPITVRHLESILRIAESFAKMRLSEFVSSYDLDRAIKVVVDSFVDAQKVSVRRQLRRSFAIYTLGH F38FF682581B0EC0 F38FF682581B0EC0 psf1_yeast DNA replication complex GINS protein PSF1 Partner of Sld five 1 YDR013W YD8119.18 PZA208 PSF1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MYGDLGNKLVLEAKRTKQLYARSNQDVNLPMYHEDIIRNILKEVSNLRKNTEYLKEQQQLGMLDDKVAKCQYFVTLLCMERNKRCLLAYQRLRTDILDSMAWNNNGLDLMSSITFSQQDTNNLSHQEQEYLKEYCDLITDLKSGDLVDIDLSGSLVPPSDVFIDVRVLKDAGEIQTEYGVFNLIKDSQFFVRQSDVERLIQQGYLQKI 4667D8152C1AB5B4 4667D8152C1AB5B4 sld5_yeast DNA replication complex GINS protein SLD5 YDR489W SLD5 protein protein yeast Saccharomyces cerevisiae Baker's yeast MDINIDDILAELDKETTAVDSTKITQGSSSTTHRDANTIVGSSLDLNDKTQIYVSPQQDFSDLMKSWKNERCSPELLPYPHQLMKRLLNRISMQSQLIENISMGFLDMQNASNANPPMPNESKLPLLCMETELERLKFVIRSYIRCRLSKIDKFSLYLRQLNEDENSLISLTDLLSKDEIKYHDTHSLIWLKLVNDSILKYMPEELQAINDTEGSVNMIDEPDWNKFVFIHVNGPPDGKWNEDPLLQENEFGKPCYTVTIPDLKEEVELTIGSIYVMRYEVIRDLLRDDKVALI 7EC516ADAD9900DD 7EC516ADAD9900DD mcm10_yeast Minichromosome maintenance protein 10 YIL150C MCM10 DNA43 Protein DNA43 protein protein yeast Saccharomyces cerevisiae Baker's yeast MNDPREILAVDPYNNITSDEEDEQAIARELEFMERKRQALVERLKRKQEFKKPQDPNFEAIEVPQSPTKNRVKVGSHNATQQGTKFEGSNINEVRLSQLQQQPKPPASTTTYFMEKFQNAKKNEDKQIAKFESMMNARVHTFSTDEKKYVPIITNELESFSNLWVKKRYIPEDDLKRALHEIKILRLGKLFAKIRPPKFQEPEYANWATVGLISHKSDIKFTSSEKPVKFFMFTITDFQHTLDVYIFGKKGVERYYNLRLGDVIAILNPEVLPWRPSGRGNFIKSFNLRISHDFKCILEIGSSRDLGWCPIVNKKTHKKCGSPINISLHKCCDYHREVQFRGTSAKRIELNGGYALGAPTKVDSQPSLYKAKGENGFNIIKGTRKRLSEEEERLKKSSHNFTNSNSAKAFFDEKFQNPDMLANLDNKRRKIIETKKSTALSRELGKIMRRRESSGLEDKSVGERQKMKRTTESALQTGLIQRLGFDPTHGKISQVLKSSVSGSEPKNNLLGKKKTVINDLLHYKKEKVILAPSKNEWFKKRSHREEVWQKHFGSKETKETSDGSASDLEII D32CFF4A94FD9B2A ctf4_yeast DNA polymerase alpha-binding protein CTF4 CHL15 POB1 YPR135W P9659.7 Chromosome transmission fidelity protein 4 Protein POB1 Chromosome replication protein CHL15 protein protein yeast Saccharomyces cerevisiae Baker's yeast MVSVIDKLVFDFGGKTLVSLAPDNNTLCVANKNGLTKILKTNNPEEEPETLDSSKLVSSIKCYSNSHFLMTTMQGDALRYNIDSSQEELLARFALPLRDCCVIHSGKMAVFGGDDLELILLELDDETHKKHAIKIDEQVSQISYNSQMNILAVSMINGKVQIFSLTSTIPNKVHELNDYIVANSYDDTHRDKILSNMMDDIDKDNDNDLSETADPDENNVADPEFCAANRICTRVAWHPKGLHFALPCADDTVKIFSIKGYSLQKTLSTNLSSTKAHFIDLQFDPLRGTYIAAVDLNNKLTVWNWETSEIHYTREFKRKITNIAWKIQADSKTLDLVLGTWSGSIAIVQNLAESVVSNIPDQSVAESSTKHGLFVDSESDLENLEGNDDINKSDKLFSDITQEANAEDVFTQTHDGPSGLSEKRKYNFEDEEDFIDDDDGAGYISGKKPHNEHSYSRVHKTHSFPISLANTGKFRYMPFSPAGTPFGFTDRRYLTMNEVGYVSTVKNSEQYSITVSFFDVGRFREYHFEDLFGYDLCFLNEKGTLFGQSKTGQIQYRPHDSIHSNWTKIIPLQAGERITSVAATPVRVIVGTSLGYFRSFNQFGVPFAVEKTSPIVALTAQNYRVFSVHYSQFHGLSYSLSELGTSSKRYYKRECPLPMSLPNINSDMKKDANLDYYNFNPMGIKSLFFSSYGDPCIFGSDNTLLLLSKWRSPEESKWLPILDSNMEIWKMSGGKETTDIHVWPLALAYDTLNCILVKGKHIWPEFPLPLPSEMEIRMPVFVKSKLLEENKAILNKKNEIGADTEAEEGEEDKEIQIPVSMAAEEEYLRSKVLSELLTDTLENDGEMYGNENEVLAALNGAYDKALLRLFASACSDQNVEKALSLAHELKQDRALTAAVKISERAELPSLVKKINNIREARYEQQLK E2ABBB244B5F8B4D E2ABBB244B5F8B4D psf2_yeast DNA replication complex GINS protein PSF2 PSF2 YJL072C J1086 Partner of Sld five 2 HRF213 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSLPAHLQQTFSPEEIQFIVENEPIKIFPRITTRQKIRGDDRGTGNHTRWQLITTDDKALNNMVAMRSTEVVLWIALLLKQQSKCSIVAPQWLTTKELDRKIQYEKTHPDRFSELPWNWLVLARILFNKAKDDFHDPIHELRGKIQDLREIRQIKVLKGLKYLNESHLQLDNLSLLEINELRPFITEIMDKLREIHTASLTAGTENDEEEFNI C8B52C0462A37A3A C8B52C0462A37A3A rfa1_yeast Replication factor A protein 1 RFA1 BUF2 YAR007C FUN3 Replication protein A 69 kDa DNA-binding subunit Single-stranded DNA-binding protein DNA-binding protein BUF2 RPA1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSVQLSRGDFHSIFTNKQRYDNPTGGVYQVYNTRKSDGANSNRKNLIMISDGIYHMKALLRNQAASKFQSMELQRGDIIRVIIAEPAIVRERKKYVLLVDDFELVQSRADMVNQTSTFLDNYFSEHPNETLKDEDITDSGNVANQTNASNAGVPDMLHSNSNLNANERKFANENPNSQKTRPIFAIEQLSPYQNVWTIKARVSYKGEIKTWHNQRGDGKLFNVNFLDTSGEIRATAFNDFATKFNEILQEGKVYYVSKAKLQPAKPQFTNLTHPYELNLDRDTVIEECFDESNVPKTHFNFIKLDAIQNQEVNSNVDVLGIIQTINPHFELTSRAGKKFDRRDITIVDDSGFSISVGLWNQQALDFNLPEGSVAAIKGVRVTDFGGKSLSMGFSSTLIPNPEIPEAYALKGWYDSKGRNANFITLKQEPGMGGQSAASLTKFIAQRITIARAQAENLGRSEKGDFFSVKAAISFLKVDNFAYPACSNENCNKKVLEQPDGTWRCEKCDTNNARPNWRYILTISIIDETNQLWLTLFDDQAKQLLGVDANTLMSLKEEDPNEFTKITQSIQMNEYDFRIRAREDTYNDQSRIRYTVANLHSLNYRAEADYLADELSKALLA 7EB8DFA6910EF8A8 7EB8DFA6910EF8A8 cdc54-cdc45-1 2 Mcm3 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level CSM3 11 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level POB3 9 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Cdc45 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm6 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm2 12 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm5 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm4 3 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Mcm7 8 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Psf1 13 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level MCM10 15 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association mint Using our standard extract conditions (see Materials and methods), the loaded Mcm2-7 complex was removed during the centrifugation steps together with almost all of the genomic DNA, whereas the unloaded Mcm2-7 complex was unstable and broke into several subcomplexes, so that isolation of the Mcm4 subunit led to efficient co-immunoprecipitation of Mcm6 but not Mcm2-3-5-7 (Figure 1B, samples 1 and 3). To isolate the unloaded Mcm2-7 complex in a stable form, we made extracts in the presence of ATP (Evrin et al, 2009; Remus et al, 2009). Under such conditions, Mcm4 associated efficiently with Mcm2-3-5-6-7 (Figure 1B, sample 4). In contrast, we found that the loaded Mcm2-7 complex was equally stable whether or not ATP was added to the extract (FvD and KL, unpublished observations), and we were thus able to isolate specifically the loaded Mcm2-7 complex by prior digestion of chromosomal DNA in extracts lacking ATP (Figure 1B, sample 2). Strikingly, Mcm10 only co-purified with the Mcm2-7 complex that had been released from chromatin (Figure 1B, compare samples 2 and 4), and we confirmed that this was not due to ATP inhibiting the interaction of Mcm10 with the unloaded complex (FvD and KL, unpublished observations). To test directly whether the association of Mcm10 with loaded Mcm2-7 in our cell extracts was independent of CDK, which is not active during G1-phase, we used the heatinducible degron (Dohmen et al, 1994; Labib et al, 2000) to regulate the level of Cdc28, the budding yeast CDK. We grew control and cdc28-td cells at 241C and then synchronised the cultures in G2-M-phase by addition of nocodazole. Under these conditions, high CDK activity blocks loading of the Mcm2-7 helicase at origins of DNA replication (Dahmann et al, 1995; Tanaka et al, 1997), so that after digesting chromatin in cell extracts lacking ATP, Mcm4 associated efficiently with Mcm6 but not with Mcm2-3-5-7 or Mcm10 (Figure 1C, IPs of Mcm4 at 241C). Following inactivation of Cdc28-td at 371C, however, reloading of Mcm2-7 was reflected by the stable association of Mcm4 with the other Mcm2-7 proteins after chromatin digestion, and Mcm10 co-immunoprecipitated with the loaded Mcm2-7 proteins as predicted (Figure 1C, cdc28-td at 371C).We also confirmed that inactivation of Cdc28-td during G1-phase blocked budding, DNA replication and the stable association of Mcm2-7 with other components of the Replisome Progression Complex such as Cdc45 and GINS, but inactivation of Cdc28-td did not affect the association of Mcm2-7 with Mcm10 in G1-phase or early S-phase (Figure 1D). These findings indicated that Mcm10 could associate with the loaded Mcm2-7 complex in the absence of CDK activity, under these experimental conditions. To test whether the association of Mcm10 with loaded Mcm2-7 was also independent of Cdc7 kinase, we synchronised control and cdc7-1 cells in G2-M-phase with nocodazole, before shifting to 371C. Cells were then washed into fresh medium containing mating pheromone so that they passed through mitosis and arrested in G1-phase of the subsequent cell cycle. Mcm10 still associated with the loaded Mcm2-7 complex in both strains, despite the fact that the subsequent round of replication was blocked in the absence of Cdc7 activity (Figure 1E) f1b f1c f1d f1e cdc54-cdc45-2 2 SLD5 14 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm6 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Ctf4 16 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level MCM10 15 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Psf1 13 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Cdc45 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Pol1 10 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Mcm5 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm4 3 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association f2d homomint mint Moreover, Mcm4 associated with Mcm2-7 proteins and Mcm10 during both G1 and S-phase, but with replisome components only during S-phase (Figure 2D). pol1-cdc45 2 Psf1 13 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Cdc45 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Pol1 10 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Mcm5 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level SLD5 14 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Ctf4 16 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association f2c homomint mint Despite its robust interaction with loaded Mcm2-7 proteins, Mcm10 did not co-purify from extracts of S-phase cells with replisome components such as Cdc45 and GINS, or with Ctf4 and the Pol1 catalytic subunit of DNA polymerase alpha. We confirmed that polymerase alpha associated with replisome material in yeast cell extracts under identical conditions (Figure 2C). mcm10-mcm2-3 2 Mcm5 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm2 12 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level MCM10 15 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Mcm6 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association f2b homomint mint Mcm10 interacted with loaded Mcm2-7 proteins in G1-phase (Figure 2B, 0 min). Upon release from G1-arrest, the complex of Mcm10 with Mcm2-7 diminished by the middle of S-phase and disappeared afterwards, reflecting the displacement of Mcm2-7 from chromatin, before reappearing when cells were blocked in the subsequent G1-phase by readdition of mating pheromone to the cell culture (Figure 2B, 90 min). cdc54-cdc45-3 2 Cdc45 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level SLD5 14 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level CSM3 11 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Ctf4 16 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm6 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm5 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm4 3 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association f4b mint As before, hyperphosphorylation of Rad53 was greatly defective when cells entered S-phase in the presence of hydroxyurea following inactivation of Mcm10-1td, in comparison with control cells (Figure 4A). Strikingly, however, Mcm4 associated not just with the other Mcm2-7 proteins but also with Cdc45, GINS, Ctf4 and Csm3 (Figure 4B), indicating that assembly of the Replisome Progression Complex could still occur in the absence of Mcm10. sld5-cdc45-3 2 Psf1 13 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level SLD5 14 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell tap tagged tap tagged calmodulin binding peptide plus protein a tag CBP-ProtA tagged CBP-ProtA tagged tap tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Cdc45 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm5 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Rfa1 18 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level association association f5b homomint mint Rfa1 co-immunoprecipitated with GINS from extracts of crosslinked control cells, reflecting the assembly of DNA replication forks containing ssDNA, Rfa1 was not detected in the analogous material isolated from mcm10-1td, despite the association of GINS with Cdc45 and Mcm2-7 (Figure 5B). cdc54-cdc45-4 2 Mcm2 12 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Psf1 13 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Psf2 17 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level SLD5 14 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm5 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Cdc45 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Mcm4 3 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association f4c homomint mint To exclude the possibility that Cdc45 and GINS only formed a very unstable complex with the loaded Mcm2-7 helicase in the absence of Mcm10, we repeated the above experiment but released cells into S-phase at 371C without hydroxyurea, and then made cell extracts containing either 100 or 700mM potassium acetate. In both cases, the interaction of GINS and Cdc45 with Mcm2-7 was comparable in extracts of control or mcm10-1td cells (Figure 4C), reflecting the inherent stability of the Cdc45 MCM GINS helicase assembly within the Replisome Progression Complex (Gambus et al, 2006). mcm10-mcm6-2 1 Mcm6 5 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell binding site binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain Winged-helix domain fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Mcm10 15 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association sf3 mint