IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ simonetta-2009-1 The mechanism of ATP-dependent primer-template recognition by a clamp loader complex. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Simonetta KR., Kazmirski SL., Goedken ER., Cantor AJ., Kelch BA., McNally R., Seyedin SN., Makino DL., O'Donnell M., Kuriyan J. kuriyan@berkeley.edu Cell (0092-8674) 2009 Only protein-protein interactions 2 imex curation simonetta-2009-2 The mechanism of ATP-dependent primer-template recognition by a clamp loader complex. in vitro In vitro fps fluorescence polarization spectroscopy Fluorescence anisotropy FPS FPS Fluorescence anisotropy fps predetermined predetermined participant predetermined Simonetta KR., Kazmirski SL., Goedken ER., Cantor AJ., Kelch BA., McNally R., Seyedin SN., Makino DL., O'Donnell M., Kuriyan J. kuriyan@berkeley.edu Cell (0092-8674) 2009 Only protein-protein interactions 2 imex curation simonetta-2009-3 The mechanism of ATP-dependent primer-template recognition by a clamp loader complex. in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Simonetta KR., Kazmirski SL., Goedken ER., Cantor AJ., Kelch BA., McNally R., Seyedin SN., Makino DL., O'Donnell M., Kuriyan J. kuriyan@berkeley.edu Cell (0092-8674) 2009 Only protein-protein interactions 2 imex curation adp adenosine 5'-(trihydrogen diphosphate) (ADP) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) trifluoroberyllate(1-) trifluoroberyllate(1−) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) a widely used substitute for the γ-phosphate of ATP ctggcctata CTGGCCTATA dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid chemical synthesis Chemical synthesis (Chemical synthesis) CTGGCCTATA ttttttataggccag TTTTTTATAGGCCAG dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid chemical synthesis Chemical synthesis (Chemical synthesis) TTTTTTATAGGCCAG holb_ecoli DNA polymerase III subunit delta' holB b1099 JW1085 protein protein ecoli Escherichia coli (strain K12) MRWYPWLRPDFEKLVASYQAGRGHHALLIQALPGMGDDALIYALSRYLLCQQPQGHKSCGHCRGCQLMQAGTHPDYYTLAPEKGKNTLGVDAVREVTEKLNEHARLGGAKVVWVTDAALLTDAAANALLKTLEEPPAETWFFLATREPERLLATLRSRCRLHYLAPPPEQYAVTWLSREVTMSQDALLAALRLSAGSPGAALALFQGDNWQARETLCQALAYSVPSGDWYSLLAALNHEQAPARLHWLATLLMDALKRHHGAAQVTNVDVPGLVAELANHLSPSRLQAILGDVCHIREQLMSVTGINRELLITDLLLRIEHYLQPGVVLPVPHL dpo3x_ecoli DNA polymerase III subunit tau dnaX dnaZ dnaZX b0470 JW0459 DNA polymerase III subunit gamma protein protein ecoli Escherichia coli (strain K12) MSYQVLARKWRPQTFADVVGQEHVLTALANGLSLGRIHHAYLFSGTRGVGKTSIARLLAKGLNCETGITATPCGVCDNCREIEQGRFVDLIEIDAASRTKVEDTRDLLDNVQYAPARGRFKVYLIDEVHMLSRHSFNALLKTLEEPPEHVKFLLATTDPQKLPVTILSRCLQFHLKALDVEQIRHQLEHILNEEHIAHEPRALQLLARAAEGSLRDALSLTDQAIASGDGQVSTQAVSAMLGTLDDDQALSLVEAMVEANGERVMALINEAAARGIEWEALLVEMLGLLHRIAMVQLSPAALGNDMAAIELRMRELARTIPPTDIQLYYQTLLIGRKELPYAPDRRMGVEMTLLRALAFHPRMPLPEPEVPRQSFAPVAPTAVMTPTQVPPQPQSAPQQAPTVPLPETTSQVLAARQQLQRVQGATKAKKSEPAAATRARPVNNAALERLASVTDRVQARPVPSALEKAPAKKEAYRWKATTPVMQQKEVVATPKALKKALEHEKTPELAAKLAAEAIERDPWAAQVSQLSLPKLVEQVALNAWKEESDNAVCLHLRSSQRHLNNRGAQQKLAEALSMLKGSTVELTIVEDDNPAVRTPLEWRQAIYEEKLAQARESIIADNNIQTLRRFFDAELDEESIRPI hola_ecoli DNA polymerase III subunit delta b0640 holA JW0635 protein protein ecoli Escherichia coli (strain K12) MIRLYPEQLRAQLNEGLRAAYLLLGNDPLLLQESQDAVRQVAAAQGFEEHHTFSIDPNTDWNAIFSLCQAMSLFASRQTLLLLLPENGPNAAINEQLLTLTGLLHDDLLLIVRGNKLSKAQENAAWFTALANRSVQVTCQTPEQAQLPRWVAARAKQLNLELDDAANQVLCYCYEGNLLALAQALERLSLLWPDGKLTLPRVEQAVNDAAHFTPFHWVDALLMGKSKRALHILQQLRLEGSEPVILLRTLQRELLLLVNLKRQSAHTPLRALFDKHRVWQNRRGMMGEALNRLSQTQLRQAVQLLTRTELTLKQDYGQSVWAELEGLSLLLCHKPLADVFIDG 87239A678FE16BE2 hold_ecoli DNA polymerase III subunit psi holD b4372 JW4334 protein protein ecoli Escherichia coli (strain K12) MTSRRDWQLQQLGITQWSLRRPGALQGEIAIAIPAHVRLVMVANDLPALTDPLVSDVLRALTVSPDQVLQLTPEKIAMLPQGSHCNSWRLGTDEPLSLEGAQVASPALTDLRANPTARAALWQQICTYEHDFFPRND gggacgcacgcggcattcaa GGGACGCACGCGGCATTCAAGGACTTAGGTCTGTATT dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid chemical synthesis Chemical synthesis (Chemical synthesis) GGGACGCACGCGGCATTCAAGGACTTAGGTCTGTATT ttgtgggtagataaatacag TTGTGGGTAGATAAATACAGACCTAAGTCCTTGAATGCCGCGTGCGTCCC dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid chemical synthesis Chemical synthesis (Chemical synthesis) TTGTGGGTAGATAAATACAGACCTAAGTCCTTGAATGCCGCGTGCGTCCC hola-dpo3x-1 Crystal Structure of the Ecoli Clamp Loader Bound to Primer-Template DNA 827799 n/a 827802 unspecified role unspecified role neutral component neutral component n/a 827803 unspecified role unspecified role neutral component neutral component n/a 827804 unspecified role unspecified role neutral component neutral component n/a 827805 unspecified role unspecified role neutral component neutral component n/a 827806 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827807 unspecified role unspecified role neutral component neutral component n-terminal 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 false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 827808 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association false false false 3.4A R-factor: 22.34%, Free R-factor: 26.04% Supp. Table 4 hola-dpo3x-2 Crystal Structure of a Mutant (gammaT157A) E. coli Clamp Loader Bound to Primer-Template DNA 827799 n/a 827802 unspecified role unspecified role neutral component neutral component n/a 827807 unspecified role unspecified role neutral component neutral component n-terminal 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 false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false P06710:p.Thr157Ala mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 827804 unspecified role unspecified role neutral component neutral component n/a 827808 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827806 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827805 unspecified role unspecified role neutral component neutral component n/a 827803 unspecified role unspecified role neutral component neutral component physical association physical association false false false 3.25A R-factor: 22.6%, Free R-factor: 26.3% The Thr157Ala mutation of P06710 prevents ATP hydrolysis. Supp. Table 4 hola-dpo3x-3 Crystal Structure of the Ecoli Clamp Loader 827799 n/a 827808 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827806 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827807 unspecified role unspecified role neutral component neutral component n-terminal 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 false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) physical association physical association false false false 3.9A R-factor: 35.93%, Free R-factor: 36.14% Supp. Table 6 hola-dpo3x-4 Crystal Structure of the E. coli clamp loader bound to Primer-Template DNA and Psi Peptide 827799 n/a 827803 unspecified role unspecified role neutral component neutral component n/a 827808 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827805 unspecified role unspecified role neutral component neutral component n/a 827804 unspecified role unspecified role neutral component neutral component n/a 827806 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827802 unspecified role unspecified role neutral component neutral component n/a 827807 unspecified role unspecified role neutral component neutral component n-terminal 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 false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 827809 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 false chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false 3.5A R-factor: 22.34%, Free R-factor: 25.73% Supp. Table 4 dnax-hola-4 DNA binding of DNAX 827800 n/a 827810 unspecified role unspecified role neutral component neutral component n/a 827806 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827808 unspecified role unspecified role neutral component neutral component P28630:p.Arg252Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P28630:p.Arg248Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P28630:p.Lys313Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 827807 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 false ecoli Escherichia coli (strain K12) n/a 827811 unspecified role unspecified role neutral component neutral component direct interaction direct interaction false false false Figs. 4B, 6B KD for DNA binding = 1.8E-7M. Mutation of Lys 313 and Arg 248, which interact with the phosphate group of the nucleotide at the -1 position, increases the value of KD to 6.1 E-6M and 1.3 E-6M for the mutants, respectively. Mutation of Arg 252, which interacts with the phosphate group of the nucleotide at the -2 position in the overhang, had a smaller effect (KD = 7.6E-7M). pH=7.5, Temp=25C dnax-hola-5 DNA binding of DNAX 827800 n/a 827808 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827811 unspecified role unspecified role neutral component neutral component n/a 827810 unspecified role unspecified role neutral component neutral component n/a 827806 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827807 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 false ecoli Escherichia coli (strain K12) n/a 827809 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 false chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction false false false Figs. 6B The values of the KD of DNA binding are 3.8E-7M and 1.8 E-5M in the presence and absence of Psi peptide, respectively. pH=7.5, Temp=25C hola-dpo3x-5 Binding of the E. coli clamp loader to Psi Peptide 827801 n/a 827807 unspecified role unspecified role neutral component neutral component n-terminal 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 false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 827809 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 false P28632:p.Trp17Ser mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false chemical synthesis Chemical synthesis (Chemical synthesis) n/a 827806 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 827808 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association false false false Fig. 6A Stoichiometry of binding of clamp loader complex to holD peptide is 1:1 Kd=7E-9M (clamp loader binding to holD peptide). pH=7.5, Temp=25C