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/ A novel nuclear protein, MGC5306 interacts with DNA polymerase beta and has a potential role in cellular phenotype A novel nuclear protein, MGC5306 interacts with DNA polymerase beta and has a potential role in cellular phenotype Cancer Res. (0008-5472) 2004 Wang L., Bhattacharyya N., Chelsea DM., Escobar PF., Banerjee S. Cancer - Interactions investigated in the context of cancer PMID 11467963 indicates that the recombinant DNA polBeta used is of human origin and gives the range for DNA polBeta Delta. A search for MGC5306 in NCBI Entrez found CAG33650 containing a reference to the TrEMBL entry Q6I9Y6, which corresponds to primary UniProtKB accession number Q9H5J8. banerjs@ccf.org 13-Nov-2007: Contacted by JYOTI. HeLa cell cDNA library davet human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Wang L., Bhattacharyya N., Chelsea DM., Escobar PF., Banerjee S. Cancer - Interactions investigated in the context of cancer PMID 11467963 indicates that the recombinant DNA polBeta used is of human origin and gives the range for DNA polBeta Delta. A search for MGC5306 in NCBI Entrez found CAG33650 containing a reference to the TrEMBL entry Q6I9Y6, which corresponds to primary UniProtKB accession number Q9H5J8. banerjs@ccf.org Accepted 2007-NOV-24 by DAVET. 13-Nov-2007: Contacted by JYOTI. HeLa cell cDNA library Cancer Res. (0008-5472) 2004 davet A novel nuclear protein, MGC5306 interacts with DNA polymerase beta and has a potential role in cellular phenotype A novel nuclear protein, MGC5306 interacts with DNA polymerase beta and has a potential role in cellular phenotype Cancer Res. (0008-5472) 2004 Wang L., Bhattacharyya N., Chelsea DM., Escobar PF., Banerjee S. Cancer - Interactions investigated in the context of cancer PMID 11467963 indicates that the recombinant DNA polBeta used is of human origin and gives the range for DNA polBeta Delta. A search for MGC5306 in NCBI Entrez found CAG33650 containing a reference to the TrEMBL entry Q6I9Y6, which corresponds to primary UniProtKB accession number Q9H5J8. banerjs@ccf.org 13-Nov-2007: Contacted by JYOTI. HeLa cell cDNA library davet human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. fluorescence imaging fluorescence microscopy fluorescence imaging tag visualisation tag visualisation Wang L., Bhattacharyya N., Chelsea DM., Escobar PF., Banerjee S. Cancer - Interactions investigated in the context of cancer PMID 11467963 indicates that the recombinant DNA polBeta used is of human origin and gives the range for DNA polBeta Delta. A search for MGC5306 in NCBI Entrez found CAG33650 containing a reference to the TrEMBL entry Q6I9Y6, which corresponds to primary UniProtKB accession number Q9H5J8. banerjs@ccf.org Accepted 2007-NOV-24 by DAVET. 13-Nov-2007: Contacted by JYOTI. HeLa cell cDNA library Cancer Res. (0008-5472) 2004 davet A novel nuclear protein, MGC5306 interacts with DNA polymerase beta and has a potential role in cellular phenotype A novel nuclear protein, MGC5306 interacts with DNA polymerase beta and has a potential role in cellular phenotype Cancer Res. (0008-5472) 2004 Wang L., Bhattacharyya N., Chelsea DM., Escobar PF., Banerjee S. Cancer - Interactions investigated in the context of cancer PMID 11467963 indicates that the recombinant DNA polBeta used is of human origin and gives the range for DNA polBeta Delta. A search for MGC5306 in NCBI Entrez found CAG33650 containing a reference to the TrEMBL entry Q6I9Y6, which corresponds to primary UniProtKB accession number Q9H5J8. banerjs@ccf.org 13-Nov-2007: Contacted by JYOTI. HeLa cell cDNA library davet 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 Wang L., Bhattacharyya N., Chelsea DM., Escobar PF., Banerjee S. Cancer - Interactions investigated in the context of cancer PMID 11467963 indicates that the recombinant DNA polBeta used is of human origin and gives the range for DNA polBeta Delta. A search for MGC5306 in NCBI Entrez found CAG33650 containing a reference to the TrEMBL entry Q6I9Y6, which corresponds to primary UniProtKB accession number Q9H5J8. banerjs@ccf.org Accepted 2007-NOV-24 by DAVET. HeLa cell cDNA library Cancer Res. (0008-5472) 2004 davet taf1d_human TATA box-binding protein-associated factor RNA polymerase I subunit D RNA polymerase I-specific TBP-associated factor 41 kDa TAF1D JOSD3 TATA box-binding protein-associated factor 1D Transcription initiation factor SL1/TIF-IB subunit D protein protein human Homo sapiens Human MDKSGIDSLDHVTSDAVELANRSDNSSDSSLFKTQCIPYSPKGEKRNPIRKFVRTPESVHASDSSSDSSFEPIPLTIKAIFERFKNRKKRYKKKKKRRYQPTGRPRGRPEGRRNPIYSLIDKKKQFRSRGSGFPFLESENEKNAPWRKILTFEQAVARGFFNYIEKLKYEHHLKESLKQMNVGEDLENEDFDSRRYKFLDDDGSISPIEESTAEDEDATHLEDNECDIKLAGDSFIVSSEFPVRLSVYLEEEDITEEAALSKKRATKAKNTGQRGLKM 4BA37C73BB387446 dpolb_human DNA polymerase beta POLB protein protein human Homo sapiens Human MSKRKAPQETLNGGITDMLTELANFEKNVSQAIHKYNAYRKAASVIAKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKIRQDDTSSSINFLTRVSGIGPSAARKFVDEGIKTLEDLRKNEDKLNHHQRIGLKYFGDFEKRIPREEMLQMQDIVLNEVKKVDSEYIATVCGSFRRGAESSGDMDVLLTHPSFTSESTKQPKLLHQVVEQLQKVHFITDTLSKGETKFMGVCQLPSKNDEKEYPHRRIDIRLIPKDQYYCGVLYFTGSDIFNKNMRAHALEKGFTINEYTIRPLGVTGVAGEPLPVDSEKDIFDYIQWKYREPKDRSE FF9A6D0E6F9A9487 xrcc1_human DNA repair protein XRCC1 XRCC1 X-ray repair cross-complementing protein 1 protein protein human Homo sapiens Human MPEIRLRHVVSCSSQDSTHCAENLLKADTYRKWRAAKAGEKTISVVLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGAGEQDYEVLLVTSSFMSPSESRSGSNPNRVRMFGPDKLVRAAAEKRWDRVKIVCSQPYSKDSPFGLSFVRFHSPPDKDEAEAPSQKVTVTKLGQFRVKEEDESANSLRPGALFFSRINKTSPVTASDPAGPSYAAATLQASSAASSASPVSRAIGSTSKPQESPKGKRKLDLNQEEKKTPSKPPAQLSPSVPKRPKLPAPTRTPATAPVPARAQGAVTGKPRGEGTEPRRPRAGPEELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTRDSTHLICAFANTPKYSQVLGLGGRIVRKEWVLDCHRMRRRLPSRRYLMAGPGSSSEEDEASHSGGSGDEAPKLPQKQPQTKTKPTQAAGPSSPQKPPTPEETKAASPVLQEDIDIEGVQSEGQDNGAEDSGDTEDELRRVAEQKEHRLPPGQEENGEDPYAGSTDENTDSEEHQEPPDLPVPELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDNMSDRVQFVITAQEWDPSFEEALMDNPSLAFVRPRWIYSCNEKQKLLPHQLYGVVPQA DIP protein P18887 original sequence version: 120 DIP protein P18887 original sequence version: 161 30DB3321234DBFC2 polb-josd3 3 5 unspecified role unspecified role bait bait region DNA polBeta Delta sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 4 unspecified role unspecified role prey prey physical association physical association A novel protein MGC5306 has been identified in yeast-two-hybrid analysis by screening a HeLa cDNA library with a truncated DNA polymerase(pol) as bait. 1 A polb-xrcc1 3 5 unspecified role unspecified role bait bait region DNA polBeta Delta sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role prey prey physical association physical association galactosidase activity was detected in the colony with XRCC1 shown in Fig. 1A, Lane 2. XRCC1, a known nuclear repair protein interacts with pol 1 A josd3-polb 1 5 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association To confirm additionally the interaction between MGC5306 and pol, we took advantage of GFP-tagged MGC5306 and analyzed by co-immunoprecipitation-Western blot analysis. A 39-kDa pol protein was identified in the immunoprecipitated product (Fig. 1B, bottom panel, Lane 1) when anti-GFP antibody was used to pull down the complex and Western blotting with anti-pol antibody, indicating the interaction of MGC5306 and endogenous WT-pol 1 B Q9H5J8 transfection josd3-polb-1 1 4 unspecified role unspecified role bait bait region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 5 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain physical association physical association To confirm additionally the interaction between MGC5306 and pol, we took advantage of GFP-tagged MGC5306 and analyzed by co-immunoprecipitation-Western blot analysis. A 39-kDa pol protein was identified in the immunoprecipitated product (Fig. 1B, bottom panel, Lane 1) when anti-GFP antibody was used to pull down the complex and Western blotting with anti-pol antibody, indicating the interaction of MGC5306 and endogenous WT-pol 1 B Q9H5J8; P06746 transfection polb-josd3-1 2 5 unspecified role unspecified role neutral component neutral component region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 4 unspecified role unspecified role neutral component neutral component colocalization colocalization we determined whether in nuclei, MGC5306 colocalizes with pol. The top right panel in Fig. 2B shows Texas Red-stained MGC5306 in nuclei of 293 cells and green fluorescence emitted from nuclei demonstrates distinct nuclear localization of pol (Fig. 2B, top left panel). Upon overlapping of both, the yellow stain (Fig. 2B, bottom left panel) in the nuclei is clearly visualized, indicating both proteins reside in nuclei and in close physical distance. 2 B Colocalization occurs in the nuclei of HEK293 cells.