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/ Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Nature (0028-0836) 2012 imex curation Armstrong A.A., Mohideen F., Lima C.D. According to Lucasz this does not yet have an ImeX ID - (presumably publication reserved as I cannot assign an ID), -they are waiting for a Kd reporting problem to be resolved (for how long?) ..... Although PDB and PDBe annotate the used Smt3 (yeast SUMO) construct as spanning residues 20 to 98, the authors describe their construct as Ndelta18 Smt3 with a Lys19Arg mutation. Details for the construct are scarce and the GST-tagged version is not described in the materials section. Based on the available information, it was decided to use Smt3 region 19-98 to define the construct used in these experiments. limac@mskcc.org 21-Jul-2014: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down predetermined predetermined participant predetermined Armstrong A.A., Mohideen F., Lima C.D. According to Lucasz this does not yet have an ImeX ID - (presumably publication reserved as I cannot assign an ID), -they are waiting for a Kd reporting problem to be resolved (for how long?) ..... Although PDB and PDBe annotate the used Smt3 (yeast SUMO) construct as spanning residues 20 to 98, the authors describe their construct as Ndelta18 Smt3 with a Lys19Arg mutation. Details for the construct are scarce and the GST-tagged version is not described in the materials section. Based on the available information, it was decided to use Smt3 region 19-98 to define the construct used in these experiments. limac@mskcc.org Accepted 2014-JUN-30 by MGT Nature (0028-0836) 2012 imex curation Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Nature (0028-0836) 2012 imex curation Armstrong A.A., Mohideen F., Lima C.D. According to Lucasz this does not yet have an ImeX ID - (presumably publication reserved as I cannot assign an ID), -they are waiting for a Kd reporting problem to be resolved (for how long?) ..... Although PDB and PDBe annotate the used Smt3 (yeast SUMO) construct as spanning residues 20 to 98, the authors describe their construct as Ndelta18 Smt3 with a Lys19Arg mutation. Details for the construct are scarce and the GST-tagged version is not described in the materials section. Based on the available information, it was decided to use Smt3 region 19-98 to define the construct used in these experiments. limac@mskcc.org 21-Jul-2014: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight by staining molecular weight estimation by staining weight by staining Armstrong A.A., Mohideen F., Lima C.D. According to Lucasz this does not yet have an ImeX ID - (presumably publication reserved as I cannot assign an ID), -they are waiting for a Kd reporting problem to be resolved (for how long?) ..... Some PCNA samples were treated with N-ethylmaleimide (NEM), which chemically modifies PCNA on Cys22 and Cys81, causing it to run on gel filtration at an apparent size 3 times smaller than the PCNA trimer. Although PDB and PDBe annotate the used Smt3 (yeast SUMO) construct as spanning residues 20 to 98, the authors describe their construct as Ndelta18 Smt3 with a Lys19Arg mutation. Details for the construct are scarce and the GST-tagged version is not described in the materials section. Based on the available information, it was decided to use Smt3 region 19-98 to define the construct used in these experiments. limac@mskcc.org Accepted 2014-JUN-30 by MGT Nature (0028-0836) 2012 imex curation Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Nature (0028-0836) 2012 imex curation Armstrong A.A., Mohideen F., Lima C.D. According to Lucasz this does not yet have an ImeX ID - (presumably publication reserved as I cannot assign an ID), -they are waiting for a Kd reporting problem to be resolved (for how long?) ..... Although PDB and PDBe annotate the used Smt3 (yeast SUMO) construct as spanning residues 20 to 98, the authors describe their construct as Ndelta18 Smt3 with a Lys19Arg mutation. Details for the construct are scarce and the GST-tagged version is not described in the materials section. Based on the available information, it was decided to use Smt3 region 19-98 to define the construct used in these experiments. limac@mskcc.org 21-Jul-2014: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Armstrong A.A., Mohideen F., Lima C.D. According to Lucasz this does not yet have an ImeX ID - (presumably publication reserved as I cannot assign an ID), -they are waiting for a Kd reporting problem to be resolved (for how long?) ..... Although PDB and PDBe annotate the used Smt3 (yeast SUMO) construct as spanning residues 20 to 98, the authors describe their construct as Ndelta18 Smt3 with a Lys19Arg mutation. Details for the construct are scarce and the GST-tagged version is not described in the materials section. Based on the available information, it was decided to use Smt3 region 19-98 to define the construct used in these experiments. limac@mskcc.org Accepted 2014-JUN-30 by MGT Nature (0028-0836) 2012 imex curation Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Nature (0028-0836) 2012 imex curation Armstrong A.A., Mohideen F., Lima C.D. According to Lucasz this does not yet have an ImeX ID - (presumably publication reserved as I cannot assign an ID), -they are waiting for a Kd reporting problem to be resolved (for how long?) ..... Although PDB and PDBe annotate the used Smt3 (yeast SUMO) construct as spanning residues 20 to 98, the authors describe their construct as Ndelta18 Smt3 with a Lys19Arg mutation. Details for the construct are scarce and the GST-tagged version is not described in the materials section. Based on the available information, it was decided to use Smt3 region 19-98 to define the construct used in these experiments. limac@mskcc.org 21-Jul-2014: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro fps fluorescence polarization spectroscopy Fluorescence anisotropy FPS FPS Fluorescence anisotropy fps predetermined predetermined participant predetermined Armstrong A.A., Mohideen F., Lima C.D. According to Lucasz this does not yet have an ImeX ID - (presumably publication reserved as I cannot assign an ID), -they are waiting for a Kd reporting problem to be resolved (for how long?) ..... Some PCNA samples were treated with N-ethylmaleimide (NEM), which chemically modifies PCNA on Cys22 and Cys81, causing it to run on gel filtration at an apparent size 3 times smaller than the PCNA trimer. Some yeast SUMO (Smt3) samples were reductively methylated, though it is not clearly stated why. Although PDB and PDBe annotate the used Smt3 (yeast SUMO) construct as spanning residues 20 to 98, the authors describe their construct as Ndelta18 Smt3 with a Lys19Arg mutation. Details for the construct are scarce and the GST-tagged version is not described in the materials section. Based on the available information, it was decided to use Smt3 region 19-98 to define the construct used in these experiments. limac@mskcc.org Accepted 2014-JUN-30 by MGT Nature (0028-0836) 2012 imex curation srs2_yeast ATP-dependent DNA helicase SRS2 SRS2 HPR5 RADH YJL092W J0913 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSNNDLWLHLVSQLNTQQRAAALFDYTRGLQVIAGPGTGKTKVLTSRVAYLILHHHIHPRDIIVTTFTNKAANEMKERLQEMLRGAGVNISELLIGTFHSICLKILYRFGHLVDLQKDWRIIDEKEIDVILDDMIEKVPDQIRDYASSITRKVNLCMPSKNGDEWTIHPKLIKKQISKLKSNAILPEEYILDSNHDAALGYFYQIYQSELSKKNTLDFDDLLMYTFRLLTRVRVLSNIKHVLVDEFQDTNGIQLDLMFLFAKGNHHLSRGMTIVGDPDQSIYAFRNALAHNFLEMGRKCPIEYSTIILVENYRSSQKILNTSEILITQQNKGRQNRAPLRAQFDLDFPPVYMNFPAYFLEAPSLVRELLYLKALPNLFTFNDFAILVRQRRQIKRIESALIEHRIPYKIIRGHSFWDSKETRAMLNLLKLIFSPNDKHAILASLLYPARGLGPATGEKIKNALDTLATDVSCFQILKDISSKKIMLDIPTKGRSVIADFISMIENCQLLLQSTLLGGLSDLFDKLYELSGLKYEYLYKDGKKKNDQLEKSEPNLLNARHKNIELLKNYFLALLSKSESSDKEKNEAIKAATDEAEPIENKVITPKEYLRNFFNSLSLHSDAAEEEESESNKDAKIKREKNGFVTISTIHGAKGLEWPVVFIPGCEEGIIPCVFNDDKKDESEEDEEEDQENSKKDASPKKTRVLSVEDSIDEERRMFFVAQTRAKYLLYLSNTVTVEDVDRPRIASRFLTTDLIKAMSDSQKLFESTNSIKKLYRILNKKPPAEDDKLFSLDQLRKDYNQFIENRRERMIWQGIQMNDVYGIQLSRNKLLGSVSDFTSAADQLRLETQNSIFPQKKLIEKSRPSKINGNYAPKSRVKSPEKRYAPETTSFHSPTKKKVYAPQYVSTTNVPSRQEFHSSTGKNIPFLRREDRSITDISPRSSTRSLKGASPNKTSHMSDDLMRPSPTRKDKVTRNIHFATAGTFRIETQSNVDELHPPEYSNKSGQSLTSSEFSGFSSACSNSDQPTNLIEDINNELDLSDEELLNDISIERRRELLGSKKTKKIKPKTRNRKSKRGDKVKVEEVIDLKSEFEEDDSRNTTAAELLHNPDDTTVDNRPIISNAKFLADAAMKKTQKFSKKVKNEPASSQMDIFSQLSRAKKKSKLNNGEIIVID AE8061B114C32120 AE8061B114C32120 eco1_yeast N-acetyltransferase ECO1 ECO1 CTF7 YFR027W Establishment of cohesion protein 1 Chromosome transmission fidelity protein 7 protein protein yeast Saccharomyces cerevisiae Baker's yeast MKARKSQRKAGSKPNLIQSKLQVNNGSKSNKIVKCDKCEMSYSSTSIEDRAIHEKYHTLQLHGRKWSPNWGSIVYTERNHSRTVHLSRSTGTITPLNSSPLKKSSPSITHQEEKIVYVRPDKSNGEVRAMTEIMTLVNNELNAPHDENVIWNSTTEEKGKAFVYIRNDRAVGIIIIENLYGGNGKTSSRGRWMVYDSRRLVQNVYPDFKIGISRIWVCRTARKLGIATKLIDVARENIVYGEVIPRYQVAWSQPTDSGGKLASKYNGIMHKSGKLLLPVYI 0AF1B59BE9E71818 0AF1B59BE9E71818 pcna_yeast Proliferating cell nuclear antigen YBR0811 YBR088C POL30 protein protein yeast Saccharomyces cerevisiae Baker's yeast MLEAKFEEASLFKRIIDGFKDCVQLVNFQCKEDGIIAQAVDDSRVLLVSLEIGVEAFQEYRCDHPVTLGMDLTSLSKILRCGNNTDTLTLIADNTPDSIILLFEDTKKDRIAEYSLKLMDIDADFLKIEELQYDSTLSLPSSEFSKIVRDLSQLSDSINIMITKETIKFVADGDIGSGSVIIKPFVDMEHPETSIKLEMDQPVDLTFGAKYLLDIIKGSSLSDRVGIRLSSEAPALFQFDLKSGFLQFFLAPKFNDEE 9B56C5851621DDDC 9B56C5851621DDDC smt3_yeast Ubiquitin-like protein SMT3 SMT3 D9719.15 YDR510W protein protein yeast Saccharomyces cerevisiae Baker's yeast MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGATY A2790DE7F315E1A7 A2790DE7F315E1A7 pol30-srs2-6 2 7 unspecified role unspecified role neutral component neutral component ecobd Escherichia coli (strain B / BL21-DE3) 5 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) direct interaction direct interaction Suppl. Fig. 1 smt3-srs2-3 2 SUMO 8 unspecified role unspecified role neutral component neutral component Q12306:p.Lys19Arg mutation mutation certain certain sequence position certain certain certain sequence position certain K R Wed May 02 18:35:05 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys19arg' Tue Jun 30 17:55:23 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys19Arg' ubiquitin domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 5 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) direct interaction direct interaction Suppl. Fig. 1 pol30-smt3-1 3 8 unspecified role unspecified role neutral component neutral component ubiquitin domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q12306:p.Lys19Arg mutation mutation certain certain sequence position certain certain certain sequence position certain K R Wed May 02 16:09:35 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys19arg' Tue Jun 30 16:01:53 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys19Arg' ecobd Escherichia coli (strain B / BL21-DE3) 7 unspecified role unspecified role neutral component neutral component P15873:p.Lys127Gly mutation mutation certain certain sequence position certain certain certain sequence position certain K G Wed May 02 18:50:53 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys127gly' Tue Jun 30 16:51:24 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys127Gly' ecobd Escherichia coli (strain B / BL21-DE3) direct interaction direct interaction Suppl. Table 2 working 21.0%, free 24.9% 2.60A pol30-srs2-1 4 5 unspecified role unspecified role neutral component neutral component P12954:p.[Gln1149Glu;Ile1152Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q E certain certain sequence position certain certain certain sequence position certain I A Wed May 02 14:52:30 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln1149glu,ile1152ala' Tue Jun 30 20:21:21 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Gln1149Glu;Ile1152Ala]' P12954:p.Gln1149Glu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q E Wed May 02 19:39:43 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln1149glu' Tue Jun 30 18:14:50 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln1149Glu' region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain P12954:p.Asp1151Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain D A Wed May 02 17:08:42 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1151ala' Tue Jun 30 18:23:05 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1151Ala' P12954:p.Ile1152Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain I A Wed May 02 17:13:56 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile1152ala' Tue Jun 30 17:01:39 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile1152Ala' P12954:p.Phe1153Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F A Wed May 02 16:12:25 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe1153ala' Tue Jun 30 17:55:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe1153Ala' P12954:p.Gln1155Glu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q E Wed May 02 15:10:24 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln1155glu' Tue Jun 30 18:07:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln1155Glu' P12954:p.Leu1156Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain L A Wed May 02 19:00:33 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'leu1156ala' Tue Jun 30 17:39:20 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Leu1156Ala' P12954:p.[Ile1152Ala;Leu1156Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain I A certain certain sequence position certain certain certain sequence position certain L A Wed May 02 17:24:23 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile1152ala,leu1156ala' Tue Jun 30 17:59:13 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ile1152Ala;Leu1156Ala]' n-terminal bodipy label bodipy label n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecobd Escherichia coli (strain B / BL21-DE3) 7 unspecified role unspecified role neutral component neutral component P15873:p.Lys127Gly mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K G Wed May 02 20:00:23 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys127gly' Tue Jun 30 20:25:13 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys127Gly' P15873:p.Phe125_Ile128delinsAlaAlaAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain FLKI AAAA Thu Jul 07 12:16:07 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was phe125ala_leu126ala_lys127ala_ile128ala Fri Jul 22 10:58:59 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'phe125ala,leu126ala,lys127ala,ile128ala' Wed May 02 18:50:44 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe_leu_lys_ile125-128ala_ala_ala_ala' Tue Jun 30 15:56:38 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe125_Ile128delinsAlaAlaAlaAla' P15873:p.Lys164Arg mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K R Wed May 02 19:09:20 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys164arg' Tue Jun 30 16:54:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys164Arg' ecobd Escherichia coli (strain B / BL21-DE3) direct interaction direct interaction Fig. 1, Fig. 4a, Fig. 4c, Suppl. Fig. 2 and 3, Suppl. Table 1 A weaker interaction is observed between Srs2 (1107-1174) and the PCNA quadruple (phe125ala_leu126ala_lys127ala_ile128ala) mutant with Kd = (11.4 +/- 2.9) x 10e-6 molar. Shorter Srs2 fragments, 1107-1167 and 1137-1174, bind with Kd = (60 +/- 18) x 10e-9 molar and Kd = (573 +/- 73) x 10e-9 molar, respectively. Mutations in Srs2 (1107-1174) also weaken the interaction: gln1149glu: Kd = (0.51 +/- 0.16) x 10e-6 molar; asp1151ala: Kd = (1.67 +/- 0.45) x 10e-6 molar; ile1152ala: Kd = (10.2 +/- 4.3) x 10e-6 molar; phe1153ala: Kd = (1.48 +/- 0.46) x 10e-6 molar; gln1155glu: Kd = (5.2 +/- 1.3) x 10e-6 molar; leu1156ala: Kd = (12.4 +/- 3.0) x 10e-6 molar; ile1152ala_leu1156ala: Kd = (18.5 +/- 6.7) x 10e-6 molar; gln1149ala_ile1152ala: Kd = (14.4 +/- 3.2) x 10e-6 molar. Similar effects were observed for the shorter Srs2 fragment (1107-1167): ile1152ala: Kd = (8.7 +/- 1.9) x 10e-6 molar; leu1156ala: Kd = (6.4 +/- 1.7) x 10e-6 molar; ile1152ala_leu1156ala: Kd = (10.0 +/- 3.3) x 10e-6 molar. Binding of the PCNA quadruple (phe125ala_leu126ala_lys127ala_ile128ala) mutant was also further reduced by mutations in Srs2 (1107-1174): leu1156ala: Kd = (61 +/- 20) x 10e-6 molar; gln1149ala_ile1152ala: Kd = (53 +/- 17) x 10e-6 molar. The PCNA lys127gly mutant and the PCNA lys164arg mutant interact with Srs2 (1107-1174) with similar affinities with (Kd = (0.169 +/- 0.026) x 10e-6 molar and Kd = (0.138 +/- 0.021) x 10e-6 molar, respectively). Srs2 (1107-1174) binds NEM-treated monomeric PCNA lys127gly mutant with Kd = (1.09 +/- 0.12) x 10e-6 molar. Mutation of the Srs2 PIP-like motif or deletion of the SIM lessen interaction whereas mutations in both prevent interaction with SUMO–PCNA (Fig. 4c). smt3-srs2-1 4 5 unspecified role unspecified role neutral component neutral component P12954:p.[Ile1152Ala;Leu1156Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain I A certain certain sequence position certain certain certain sequence position certain L A Wed May 02 15:19:34 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile1152ala,leu1156ala' Tue Jun 30 17:01:18 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ile1152Ala;Leu1156Ala]' P12954:p.Leu1156Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain L A Wed May 02 18:09:57 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'leu1156ala' Tue Jun 30 19:15:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Leu1156Ala' P12954:p.[Gln1149Glu;Ile1152Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain I A certain certain sequence position certain certain certain sequence position certain Q E Fri Jul 22 11:10:58 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'ile1152ala,gln1149glu' Wed May 02 16:52:42 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln1149glu,ile1152ala' Tue Jun 30 20:25:40 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Gln1149Glu;Ile1152Ala]' P12954:p.Ile1152Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain I A Wed May 02 17:45:40 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile1152ala' Tue Jun 30 15:59:24 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile1152Ala' region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain n-terminal bodipy label bodipy label n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecobd Escherichia coli (strain B / BL21-DE3) 8 unspecified role unspecified role neutral component neutral component 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 Q12306:p.Lys19Arg mutation mutation certain certain sequence position certain certain certain sequence position certain K R Wed May 02 17:16:34 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys19arg' Tue Jun 30 17:42:20 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys19Arg' ubiquitin domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 47 52 direct interaction direct interaction Fig. 1, Fig. 4c, Suppl. Fig. 2 and 3, Suppl. Table 1 Shorter Srs2 fragments, 1107-1167 and 1137-1174, bind with Kd = (62 +/- 21) x 10e-6 molar and Kd = (3.40 +/- 0.76) x 10e-6 molar, respectively. Mutations in Srs2 (1107-1174) also weaken the interaction: leu1156ala: Kd = (1.09 +/- 0.21) x 10e-6 molar; ile1152ala_leu1156ala: Kd = (1.42 +/- 0.10) x 10e-6 molar; gln1149ala_ile1152ala: Kd = (1.27 +/- 0.17) x 10e-6 molar. Similar effects were observed for the shorter Srs2 fragment (1107-1167): ile1152ala: Kd = (85 +/- 15) x 10e-6 molar; leu1156ala: Kd = (85 +/- 61) x 10e-6 molar; ile1152ala_leu1156ala: Kd = (88 +/- 17) x 10e-6 molar. pol30-smt3-2 4 5 unspecified role unspecified role neutral component neutral component P12954:p.Asp1151Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain D A Wed May 02 17:11:09 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1151ala' Tue Jun 30 20:05:41 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1151Ala' P12954:p.Gln1155Glu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q E Wed May 02 17:33:47 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln1155glu' Tue Jun 30 20:18:05 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gln1155Glu' P12954:p.[Ile1152Ala;Leu1156Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain I A certain certain sequence position certain certain certain sequence position certain L A Wed May 02 15:49:35 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile1152ala,leu1156ala' Tue Jun 30 17:59:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ile1152Ala;Leu1156Ala]' region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain n-terminal bodipy label bodipy label n-term range n-terminal range n-term range n-term range n-terminal range n-term range P12954:p.Ile1152Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain I A Wed May 02 18:07:55 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile1152ala' Tue Jun 30 15:56:18 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile1152Ala' P12954:p.Leu1156Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain L A Wed May 02 15:49:21 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'leu1156ala' Tue Jun 30 16:22:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Leu1156Ala' P12954:p.[Gln1149Glu;Ile1152Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain Q E certain certain sequence position certain certain certain sequence position certain I A Wed May 02 19:25:39 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gln1149glu,ile1152ala' Tue Jun 30 19:13:58 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Gln1149Glu;Ile1152Ala]' ecobd Escherichia coli (strain B / BL21-DE3) 8 unspecified role unspecified role neutral component neutral component Q12306:p.Lys19Arg mutation mutation certain certain sequence position certain certain certain sequence position certain K R Wed May 02 15:59:31 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys19arg' Tue Jun 30 19:02:14 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys19Arg' ubiquitin domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 7 unspecified role unspecified role neutral component neutral component P15873:p.Phe125_Ile128delinsAlaAlaAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain FLKI AAAA Thu Jul 07 12:16:08 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was phe125ala_leu126ala_lys127ala_ile128ala Fri Jul 22 11:09:03 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'phe125ala,leu126ala,lys127ala,ile128ala' Wed May 02 19:55:00 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe_leu_lys_ile125-128ala_ala_ala_ala' Tue Jun 30 16:16:42 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe125_Ile128delinsAlaAlaAlaAla' P15873:p.Lys127Gly mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K G Wed May 02 14:26:58 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys127gly' Tue Jun 30 19:30:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys127Gly' The lys127gly mutation directs SUMO modification to lys164. P15873:p.Lys164Arg mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K R Wed May 02 17:26:49 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys164arg' Tue Jun 30 15:34:40 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys164Arg' The lys164arg mutation directs SUMO modification to lys127. P15873:p.[Val186Asp;Met188_His190delinsAlaAlaAla] mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain V D certain certain sequence position certain certain certain sequence position certain MEH AAA Thu Jul 07 12:16:39 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was val186asp_met188ala_glu189ala_his190ala Fri Jul 22 10:52:24 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'val186asp,met188ala,glu189ala,his190ala' Wed May 02 15:22:31 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'val186asp,met_glu_his188-190ala_ala_ala' Tue Jun 30 17:58:40 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Val186Asp;Met188_His190delinsAlaAlaAla]' ecobd Escherichia coli (strain B / BL21-DE3) physical association physical association Fig. 1, Fig. 4a, Fig. 4c, Suppl. Fig. 2 and 3, Suppl. Table 1 In case of reductively methylated SUMO: Srs2 (1107-1174) interacts with PCNA sumoylated on lys164 (lys127gly mutant) with Kd = (26.3 +/- 3.1) x 10e-9 molar. Srs2 (1107-1174) interacts with PCNA sumoylated on lys127 (lys164arg mutant) with Kd = (25.1 +/- 4.9) x 10e-9 molar. Srs2 (1107-1174) interacts with PCNA quadruple (phe125ala_leu126ala_lys127ala_ile128ala) mutant sumoylated on lys164 (lys127gly mutant) with Kd = (45.7 +/- 9.7) x 10e-9 molar. Shorter Srs2 fragments, 1107-1167 and 1137-1174, bind to PCNA sumoylated on lys164 (lys127gly mutant) with Kd = (89 +/- 13) x 10e-9 molar and Kd = (3.50 +/- 1.0) x 10e-9 molar, respectively. Srs2 (1107-1174) binds to NEM-treated monomeric PCNA sumoylated on lys164 (Kd = (79 +/- 18) x 10e-9 molar) or on lys127 (Kd = (14 +/- 3.9) x 10e-9 molar). The val186ala_met188ala_glu189ala_his190ala mutations in PCNA sumoylated on lys164 have no effect on binding of Srs2 (1107-1174) (Kd = (29.7 +/- 6) x 10e-9 molar). In case of non-reduced SUMO: Srs2 (1107-1174) interacts with PCNA sumoylated on lys164 (lys127gly mutant) with Kd = (11.3 +/- 3.6) x 10e-9 molar. Mutation of the Srs2 PIP-like motif or deletion of the SIM lessen interaction whereas mutations in both prevent interaction with SUMO–PCNA (Fig. 4c) smt3-srs2-2 4 5 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain n-terminal bodipy label bodipy label n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecobd Escherichia coli (strain B / BL21-DE3) 5 competitor competitor competitor neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 8 unspecified role unspecified role neutral component neutral component 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 ubiquitin domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q12306:p.Lys19Arg mutation mutation certain certain sequence position certain certain certain sequence position certain K R Wed May 02 20:12:17 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys19arg' Tue Jun 30 18:35:59 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys19Arg' ecobd Escherichia coli (strain B / BL21-DE3) physical association physical association Suppl. Fig. 2 pol30-srs2-2 4 7 unspecified role unspecified role neutral component neutral component ecobd Escherichia coli (strain B / BL21-DE3) 5 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain n-terminal bodipy label bodipy label n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecobd Escherichia coli (strain B / BL21-DE3) 5 competitor competitor competitor neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) physical association physical association Suppl. Fig. 2 pol30-smt3-3 4 7 unspecified role unspecified role neutral component neutral component P15873:p.Lys127Gly mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K G Wed May 02 15:48:31 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys127gly' Tue Jun 30 18:10:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys127Gly' The lys127gly mutation directs SUMO modification to lys164. ecobd Escherichia coli (strain B / BL21-DE3) 5 unspecified role unspecified role neutral component neutral component n-terminal bodipy label bodipy label n-term range n-terminal range n-term range n-term range n-terminal range n-term range region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 5 competitor competitor competitor neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 8 unspecified role unspecified role neutral component neutral component Q12306:p.Lys19Arg mutation mutation certain certain sequence position certain certain certain sequence position certain K R Wed May 02 19:45:18 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys19arg' Tue Jun 30 18:34:25 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys19Arg' ubiquitin domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) physical association physical association Suppl. Fig. 2, Suppl. Fig. 3 pol30-smt3-4 3 5 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 8 unspecified role unspecified role neutral component neutral component ubiquitin domain sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain Q12306:p.Lys19Arg mutation mutation certain certain sequence position certain certain certain sequence position certain K R Wed May 02 18:32:53 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys19arg' Tue Jun 30 16:53:09 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys19Arg' ecobd Escherichia coli (strain B / BL21-DE3) 7 unspecified role unspecified role neutral component neutral component P15873:p.Lys127Gly mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K G Wed May 02 19:59:20 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys127gly' Tue Jun 30 15:34:53 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys127Gly' The lys127gly mutation directs SUMO modification to lys164. ecobd Escherichia coli (strain B / BL21-DE3) physical association physical association Suppl. Table 2 working 21.6%, free 26.1% 2.90A eco1-pol30-1 1 6 unspecified role unspecified role bait bait n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecobd Escherichia coli (strain B / BL21-DE3) 7 unspecified role unspecified role prey prey P15873:p.Phe125_Ile128delinsAlaAlaAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain FLKI AAAA Thu Jul 07 12:15:50 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was phe125ala_leu126ala_lys127ala_ile128ala Fri Jul 22 11:08:28 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'phe125ala,leu126ala,lys127ala,ile128ala' Wed May 02 20:05:59 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe_leu_lys_ile125-128ala_ala_ala_ala' Tue Jun 30 18:29:59 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Phe125_Ile128delinsAlaAlaAlaAla' ecobd Escherichia coli (strain B / BL21-DE3) direct interaction direct interaction Suppl. Fig. 14 Neither the K164R or the K127G mutations, nor sumoylation of PCNA at K127 or K164 affected this interaction. pol30-1 2 7 unspecified role unspecified role neutral component neutral component ecobd Escherichia coli (strain B / BL21-DE3) direct interaction direct interaction Suppl. Fig. 7a N-ethylmaleimide (NEM) chemically modifies PCNA on Cys22 and Cys81, causing it to run on gel filtration at an apparent size 3 times smaller than the PCNA trimer. pol30-srs2-4 2 5 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 7 unspecified role unspecified role neutral component neutral component P15873:p.Lys127Gly mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K G Wed May 02 19:48:17 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys127gly' Tue Jun 30 16:55:13 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys127Gly' lys-164 sumoylated lysine sumoylated lysine certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) physical association physical association Suppl. Fig. 7b pol30-srs2-5 2 7 unspecified role unspecified role neutral component neutral component lys-164 sumoylated lysine sumoylated lysine certain certain sequence position certain certain certain sequence position certain P15873:p.Lys127Gly mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K G Wed May 02 18:32:59 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys127gly' Tue Jun 30 18:27:54 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys127Gly' ecobd Escherichia coli (strain B / BL21-DE3) 5 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) physical association physical association Suppl. Fig. 7b N-ethylmaleimide (NEM) chemically modifies PCNA on Cys22 and Cys81, causing it to run on gel filtration at an apparent size 3 times smaller than the PCNA trimer. pol30-smt3-7 3 7 unspecified role unspecified role neutral component neutral component P15873:p.Lys127Gly mutation mutation certain certain sequence position certain certain certain sequence position certain K G Wed May 02 19:59:55 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys127gly' Tue Jun 30 17:32:46 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys127Gly' ecobd Escherichia coli (strain B / BL21-DE3) 8 unspecified role unspecified role neutral component neutral component ubiquitin domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q12306:p.Lys19Arg mutation mutation certain certain sequence position certain certain certain sequence position certain K R Wed May 02 17:15:40 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys19arg' Tue Jun 30 19:25:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys19Arg' ecobd Escherichia coli (strain B / BL21-DE3) direct interaction direct interaction Suppl. Table 2 2.80A working 21.4%, free 25.4%