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/ Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Oncogene (0950-9232) 2007 imex curation Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. orchard Only protein-protein interactions human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr Accepted 2007-OCT-3 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Oncogene (0950-9232) 2007 imex curation Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. orchard Only protein-protein interactions human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr Accepted 2007-OCT-3 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Oncogene (0950-9232) 2007 imex curation Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. orchard Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr Accepted 2007-OCT-3 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Oncogene (0950-9232) 2007 imex curation Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. orchard Only protein-protein interactions human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr Accepted 2007-OCT-3 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Oncogene (0950-9232) 2007 imex curation Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr Accepted 2007-OCT-3 by ORCHARD. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Oncogene (0950-9232) 2007 imex curation Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. orchard Only protein-protein interactions human-jurkat Homo sapiens jurkat Human T cell leukemia anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr Accepted 2007-OCT-3 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Oncogene (0950-9232) 2007 imex curation Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. 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 Buchsbaum S., Morris C., Bochard V., Jalinot P. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the p110 subunit of eIF3 or RFP since the exact gene product of the interactor cannot be ascertained. pjalinot@ens-lyon.fr Accepted 2007-OCT-3 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A library of cDNAs prepared from human lymphocytes immortalized with EBV was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation int6_human Integrator complex subunit 6 INTS6 DBI1 DDX26 DDX26A DBI-1 Protein deleted in cancer 1 Protein DDX26 protein protein human Homo sapiens Human MPILLFLIDTSASMNQRSHLGTTYLDTAKGAVETFMKLRARDPASRGDRYMLVTFEEPPYAIKAGWKENHATFMNELKNLQAEGLTTLGQSLRTAFDLLNLNRLVTGIDNYGQGRNPFFLEPAIIITITDGSKLTTTSGVQDELHLPLNSPLPGSELTKEPFRWDQRLFALVLRLPGTMSVESEQLTGVPLDDSAITPMCEVTGGRSYSVCSPRMLNQCLESLVQKVQSGVVINFEKAGPDPSPVEDGQPDISRPFGSQPWHSCHKLIYVRPNPKTGVPIGHWPVPESFWPDQNSPTLPPRTSHPVVKFSCTDCEPMVIDKLPFDKYELEPSPLTQFILERKSPQTCWQVYVSNSAKYSELGHPFGYLKASTALNCVNLFVMPYNYPVLLPLLDDLFKVHKAKPTLKWRQSFESYLKTMPPYYLGPLKKAVRMMGAPNLIADSMEYGLSYSVISYLKKLSQQAKIESDRVIGSVGKKVVQETGIKVRSRSHGLSMAYRKDFQQLLQGISEDVPHRLLDLNMKEYTGFQVALLNKDLKPQTFRNAYDIPRRNLLDHLTRMRSNLLKSTRRFLKGQDEDQVHSVPIAQMGNYQEYLKQVPSPLRELDPDQPRRLHTFGNPFKLDKKGMMIDEADEFVAGPQNKHKRPGEPNMQGIPKRRRCMSPLLRGRQQNPVVNNHIGGKGPPAPTTQAQPDLIKPLPLHKISETTNDSIIHDVVENHVADQLSSDITPNAMDTEFSASSPASLLERPTNHMEALGHDHLGTNDLTVGGFLENHEEPRDKEQCAEENIPASSLNKGKKLMHCRSHEEVNTELKAQIMKEIRKPGRKYERIFTLLKHVQGSLQTRLIFLQNVIKEASRFKKRMLIEQLENFLDEIHRRANQINHINSN FCD3FD61B7767E03 ubb_human Polyubiquitin-B UBB protein protein human Homo sapiens Human MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGC 33011162F1C48BB1 mcm7_human DNA replication licensing factor MCM7 MCM7 CDC47 MCM2 CDC47 homolog P1.1-MCM3 protein protein human Homo sapiens Human MALKDYALEKEKVKKFLQEFYQDDELGKKQFKYGNQLVRLAHREQVALYVDLDDVAEDDPELVDSICENARRYAKLFADAVQELLPQYKEREVVNKDVLDVYIEHRLMMEQRSRDPGMVRSPQNQYPAELMRRFELYFQGPSSNKPRVIREVRADSVGKLVTVRGIVTRVSEVKPKMVVATYTCDQCGAETYQPIQSPTFMPLIMCPSQECQTNRSGGRLYLQTRGSRFIKFQEMKMQEHSDQVPVGNIPRSITVLVEGENTRIAQPGDHVSVTGIFLPILRTGFRQVVQGLLSETYLEAHRIVKMNKSEDDESGAGELTREELRQIAEEDFYEKLAASIAPEIYGHEDVKKALLLLLVGGVDQSPRGMKIRGNINICLMGDPGVAKSQLLSYIDRLAPRSQYTTGRGSSGVGLTAAVLRDSVSGELTLEGGALVLADQGVCCIDEFDKMAEADRTAIHEVMEQQTISIAKAGILTTLNARCSILAAANPAYGRYNPRRSLEQNIQLPAALLSRFDLLWLIQDRPDRDNDLRLAQHITYVHQHSRQPPSQFEPLDMKLMRRYIAMCREKQPMVPESLADYITAAYVEMRREAWASKDATYTSARTLLAILRLSTALARLRMVDVVEKEDVNEAIRLMEMSKDSLLGDKGQTARTQRPADVIFATVRELVSGGRSVRFSEAEQRCVSRGFTPAQFQAALDEYEELNVWQVNASRTRITFV DIP protein P33993 original sequence version: 161 DIP protein P33993 original sequence version: 124 330A1DEFAEFBFB88 ints6-mcm7 5 10 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1381855]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 720-720, sequence length 719 [EBI-1381855]515-720 8 unspecified role unspecified role bait bait physical association physical association 1 A A library of cDNAs prepared from human lymphocytes immortalized with EBV (Durfee et al., 1993) was screened by the two-hybrid method using INT6 fused to the GAL4DNA-bi nding domain as bait. This led to isolation of partial cDNAs encoding the p110 subunit of eIF3 and Rfp, as reported previously (Morris-Desbois et al., 1999), but also to clones corresponding to the C-terminal part of MCM7, the shortest one encoding the C-terminal 205 amino acids of the protein ints6-mcm7-1 4 10 unspecified role unspecified role prey prey n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 8 unspecified role unspecified role bait bait c-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association 1 C Precipitation of INT6 using the antibody to FLAG was found to bring down MCM7 and conversely INT6 was coprecipitated when MCM7 was pulled down with the antibody to the MYC epitope Q9UL03; P33993 transfection MG132 (proteasome inhibitor) treatment enhances this interaction. ints6-mcm7-2 4 10 unspecified role unspecified role bait bait n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 8 unspecified role unspecified role prey prey c-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association 1 C Precipitation of INT6 using the antibody to FLAG was found to bring down MCM7 and conversely INT6 was coprecipitated when MCM7 was pulled down with the antibody to the MYC epitope Q9UL03; P33993 transfection MG132 (proteasome inhibitor) treatment enhances this interaction. ints6-mcm7-3 6 8 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association 1 D Extracts of Jurkat (Figure 1d, lanes 1-3) and HeLa cells (Figure 1d, lanes 4-6) were immunoprecipitated with an antibody directed against INT6 or the corresponding preimmune serum. Immunoblot analysis of MCM7 revealed that the protein which appears as a double band was clearly coprecipitated with INT6 in both cell types, although slightly more efficiently in Jurkat (Figure 1d, compare lanes 2 and 5), showing that the interaction occurs between endogenous proteins. ints6-mcm7-4 2 8 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association 1 D Extracts of Jurkat (Figure 1d, lanes 1-3) and HeLa cells (Figure 1d, lanes 4-6) were immunoprecipitated with an antibody directed against INT6 or the corresponding preimmune serum. Immunoblot analysis of MCM7 revealed that the protein which appears as a double band was clearly coprecipitated with INT6 in both cell types, although slightly more efficiently in Jurkat (Figure 1d, compare lanes 2 and 5), showing that the interaction occurs between endogenous proteins. mcm7-ints6 7 10 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey region 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 the two-hybrid assay was used with MCM7 fused to the GAL4DNA binding domain as bait and INT6 deleted either on the N-terminal or C-terminal side fused to the GAL4-activation domain. At the C-terminus, removal of amino acids 395-331 led to loss of the binding of INT6 to MCM7 (Figure 1e). On the other hand, each construct deleted at the N-terminal end, up to amino acid 350, led to a positive result (Figure 1e). These data indicate that the MCM7-binding domain of INT6 is located between amino acids 351 and 395. 1 E ints6-mcm7-5 7 10 unspecified role unspecified role prey prey region 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 Similarly, various pieces of the C-terminal domain of MCM7 were associated with the GAL4-activation domain and tested by the twohybrid assay with INT6 as bait. On the N-terminal side, deletion from amino acid 579�610 led to loss of the interaction (Figure 1f), whereas at the C-terminal end removal of amino acid 595�575 impaired the interaction (Figure 1f). These results show that the part of MCM7 mediating the interaction with INT6 is located between amino acids 579 and 595, outside the MCM box. 1B & 1F mcm7-ints6-1 3 10 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role prey prey human-jurkat Homo sapiens jurkat Human T cell leukemia physical association physical association 1 G A signal corresponding to INT6 was clearly detected with beads including the MCM7 peptide, but not with those corresponding to GST alone mcm7-ints6-2 3 8 unspecified role unspecified role prey prey region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 1 H The same experiment was performed with INT6 expressed in bacteria and purified to homogeneity. As before INT6 was seen with the beads bearing the MCM7 peptide, but not with the control beads mcm7-rps27a 1 9 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait physical association physical association 2 A MCM7 was immunoprecipitated and analysed using an antibody recognizing polyubiquitylated proteins, a weak smear of bands above the position of unmodified MCM7 was observed and its intensity was reinforced by a proteasome inhibitor treatment (Figure 2a, compare lanes 2 and 4), strongly suggesting that endogenous MCM7 is polyubiquitylated and that these polyubiquitylated forms are processed by the proteasome. ints6-mcm7-6 1 9 unspecified role unspecified role bait bait n-terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range 10 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2 C immunoprecipitation using the HA antibody showed that MCM7 is polyubiquitylated, but this was not seen for MCM4 and MCM6 P33993; P62988 transfection mcm7-ints6-3 2 10 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association S1 B a low amount of MCM7 was coprecipitated with INT6 in the nucleoplasmic fraction and this was increased following proteasome inhibition (Supplementary Figure S1b, lanes 2 and 5). Using the chromatin fraction, INT6 was also found to bind MCM7, but mainly when cells were treated with MG132 This interaction occurred in the nucleoplasmic faction and the chromatin fraction. MG132 treatment (proteasome inhibition) enhances this interaction.