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 key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. Cell (0092-8674) 2007 imex curation Mortier-Barriere I., Velten M., Dupaigne P., Mirouze N., Pietrement O., McGovern S., Fichant G., Martin B., Noirot P., Le Cam E., Polard P., Claverys JP. patrice.polard@ibcg.biotoul.fr,claverys@ibcg.biotoul.fr 13-Nov-2007: Contacted by JYOTI. davet Only protein-protein interactions 5 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 Mortier-Barriere I., Velten M., Dupaigne P., Mirouze N., Pietrement O., McGovern S., Fichant G., Martin B., Noirot P., Le Cam E., Polard P., Claverys JP. patrice.polard@ibcg.biotoul.fr,claverys@ibcg.biotoul.fr Accepted 2007-NOV-25 by DAVET. 13-Nov-2007: Contacted by JYOTI. Cell (0092-8674) 2007 davet Only protein-protein interactions 5 imex curation A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. Cell (0092-8674) 2007 imex curation Mortier-Barriere I., Velten M., Dupaigne P., Mirouze N., Pietrement O., McGovern S., Fichant G., Martin B., Noirot P., Le Cam E., Polard P., Claverys JP. patrice.polard@ibcg.biotoul.fr,claverys@ibcg.biotoul.fr 13-Nov-2007: Contacted by JYOTI. davet Only protein-protein interactions 5 in vitro In vitro enzymatic study enzymatic study predetermined predetermined participant predetermined Mortier-Barriere I., Velten M., Dupaigne P., Mirouze N., Pietrement O., McGovern S., Fichant G., Martin B., Noirot P., Le Cam E., Polard P., Claverys JP. patrice.polard@ibcg.biotoul.fr,claverys@ibcg.biotoul.fr Accepted 2007-NOV-25 by DAVET. 13-Nov-2007: Contacted by JYOTI. Cell (0092-8674) 2007 davet Only protein-protein interactions 5 imex curation A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. Cell (0092-8674) 2007 imex curation Mortier-Barriere I., Velten M., Dupaigne P., Mirouze N., Pietrement O., McGovern S., Fichant G., Martin B., Noirot P., Le Cam E., Polard P., Claverys JP. patrice.polard@ibcg.biotoul.fr,claverys@ibcg.biotoul.fr 13-Nov-2007: Contacted by JYOTI. davet Only protein-protein interactions 5 strpn Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4) pull down pull down ms participant Identification by mass spectrometry ms participant Mortier-Barriere I., Velten M., Dupaigne P., Mirouze N., Pietrement O., McGovern S., Fichant G., Martin B., Noirot P., Le Cam E., Polard P., Claverys JP. patrice.polard@ibcg.biotoul.fr,claverys@ibcg.biotoul.fr Accepted 2007-NOV-25 by DAVET. 13-Nov-2007: Contacted by JYOTI. Cell (0092-8674) 2007 davet Only protein-protein interactions 5 imex curation A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. Cell (0092-8674) 2007 imex curation Mortier-Barriere I., Velten M., Dupaigne P., Mirouze N., Pietrement O., McGovern S., Fichant G., Martin B., Noirot P., Le Cam E., Polard P., Claverys JP. patrice.polard@ibcg.biotoul.fr,claverys@ibcg.biotoul.fr 13-Nov-2007: Contacted by JYOTI. davet Only protein-protein interactions 5 in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Mortier-Barriere I., Velten M., Dupaigne P., Mirouze N., Pietrement O., McGovern S., Fichant G., Martin B., Noirot P., Le Cam E., Polard P., Claverys JP. patrice.polard@ibcg.biotoul.fr,claverys@ibcg.biotoul.fr Accepted 2007-NOV-25 by DAVET. Cell (0092-8674) 2007 davet Only protein-protein interactions 5 imex curation reca_strpn Protein RecA recA SP_1940 Recombinase A protein protein strpn Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4) MAKKPKKLEEISKKFGAEREKALNDALKLIEKDFGKGSIMRLGERAEQKVQVMSSGSLALDIALGSGGYPKGRIIEIYGPESSGKTTVALHAVAQAQKEGGIAAFIDAEHALDPAYAAALGVNIDELLLSQPDSGEQGLEIAGKLIDSGAVDLVVVDSVAALVPRAEIDGDIGDSHVGLQARMMSQAMRKLGASINKTKTIAIFINQLREKVGVMFGNPETTPGGRALKFYASVRLDVRGNTQIKGTGDQKETNVGKETKIKVVKNKVAPPFKEAVVEIMYGEGISKTGELLKIASDLDIIKKAGAWYSYKDEKIGQGSENAKKYLAEHPEIFDEIDKQVRSKFGLIDGEEVSEQDTENKKDEPKKEEAVNEEVPLDLGDELEIEIEE 40F5DA5F15E1BCB3 dt90_dna 90-mer poly-dT (dT90) ss dna single stranded deoxyribonucleic acid ss DNA ss dna chemical synthesis Chemical synthesis (Chemical synthesis) TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 0F5F0E1A4B0E1A4B atp ATP Adenosine 5'-triphosphate Adenosine triphosphate ADENOSINE-5'-TRIPHOSPHATE Adephos Adetol Adynol Atipi ATP Atriphos Cardenosine Fosfobion Glucobasin H4atp Myotriphos Triadenyl Triphosphaden adenosine 5'-(tetrahydrogen triphosphate) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) 80mer_random_ssdna 80-mer oligonucleotide of random sequence ss dna single stranded deoxyribonucleic acid ss DNA ss dna chemical synthesis Chemical synthesis (Chemical synthesis) This is an 80-mer (ssDNA) of random sequence. a0a0h2uqa6_strpn SP_1266 protein protein strpn Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4) MELFMKITNYEIYKLKKSGLTNQQILKVLEYGENVDQELLLGDIADISGCRNPAVFMERYFQIDDAHLSKEFQKFPSFSILDDCYPWDLSEIYDAPVLLFYKGNLDLLKFPKVAVVGSRACSKQGAKSVEKVIQGLENELVIVSGLAKGIDTAAHMAALQNGGKTIAVIGTGLDVFYPKANKRLQDYIGNDHLVLSEYGPGEQPLKFHFPARNRIIAGLCRGVIVAEAKMRSGSLITCERAMEEGRDVFAIPGSILDGLSDGCHHLIQEGAKLVTSGQDVLAEFEF CF12DB83AE3663A2 genome_bpphx_dna Complete genome of bacteriophage phi-X174 (5386 bp ss-DNA) ss dna single stranded deoxyribonucleic acid ss DNA ss dna bpphx Enterobacteria phage phiX174 Bacteriophage phi-X174 This is the complete circular ssDNA genome of bacteriophage phi-X174 (5386 bp). reca_bacsu Protein RecA recA recE BSU16940 Recombinase A protein protein bacsu Bacillus subtilis MSDRQAALDMALKQIEKQFGKGSIMKLGEKTDTRISTVPSGSLALDTALGIGGYPRGRIIEVYGPESSGKTTVALHAIAEVQQQGGQAAFIDAEHALDPVYAQKLGVNIEELLLSQPDTGEQALEIAEALVRSGAVDIVVVDSVAALVPKAEIEGDMGDSHVGLQARLMSQALRKLSGAINKSKTIAIFINQIREKVGVMFGNPETTPGGRALKFYSSVRLEVRRAEQLKQGNDVMGNKTKIKVVKNKVAPPFRTAEVDIMYGEGISKEGEIIDLGTELDIVQKSGSWYSYEEERLGQGRENAKQFLKENKDIMLMIQEQIREHYGLDNNGVVQQQAEETQEELEFEE EE558B6354119260 dpra_bacsu DNA processing protein DprA dprA Protein Smf smf BSU16110 protein protein bacsu Bacillus subtilis MDQAAVCLTICRINQLLSPSLLLKWWKADPSMSLTSPVLQTVTRDQIKAAALKNEIEQFYPKLPRVLAAYREQGINTIPISSKQYPFWLKSIYDPPAVLFAKGDMTLLSKGRKIGIVGTRNPTAYGKQVVNHLTKEICRKGWVIVSGLASGIDGMSHAASIKAKGRTIGVIAGGFQHIYPRENLQLADHMAKHHILLSEHPPETKPQKWHFPMRNRIISGLSEGVIVVQGKEKSGSLITAYQALEQGREVFAVPGSLFDPYAGGPIKLIQQGAKAIWSAEDIFEELPERNVQYTEPF A2E369AAC4D158CB reca_ecoli Protein RecA recA lexB recH rnmB umuB tif zab b2699 Recombinase A JW2669 protein protein ecoli Escherichia coli (strain K12) MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGSLSLDIALGAGGLPMGRIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIYARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDSHMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASVRLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVKEKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSVDDSEGVAETNEDF DIP protein P0A7G6 original sequence version: 97 DIP protein P0A7G6 original sequence version: 52 DIP protein P0A7G6 original sequence version: 68 866EF8F83BE32A36 dt90_dna-q97qfo 4 9 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component direct interaction direct interaction 2 B The ability of DprA to bind oligonucleotides of various size and sequence was tested by electro mobility shift assay (EMSA). DprA interacted with a 90-mer poly-dT (dT90) The 90-mer poly-dT was labelled with 32P. This indicates that DprA binds to nonspecific ssDNA. DprA was found to render dT90 resistant to a 3->5 ssDNA exonuclease. 80mer_rando-q97qfo-1 4 9 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component direct interaction direct interaction S3 A The 80-mer oligonucleotide of random sequence was labelled with 32P. The ability of DprA to bind oligonucleotides of various size and sequence was tested by electro mobility shift assay (EMSA). DprA interacted with a 90-mer poly-dT (dT90) (Figure 2B) as well as with an 80-mer of random sequence This indicates that DprA binds to non-specific ssDNA. q97qf0-gen_bpphx 4 10 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component direct interaction direct interaction 3 A To determine whether DprA and its B. subtilis counterpart, Smf, require a free end for binding, we investigated their interaction with circular FX174 (FX) ssDNA. When increasing concentrations of the proteins were added to FX ssDNA, the mobility of ssDNA in native agarose gels decreased progressively This indicates that a free end on the substrate oligonucleotide is not necessary for DprA binding. DprA also fully protected the phiX genome from Mung Bean nuclease. smf-gen_bpphx-1 4 12 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component direct interaction direct interaction 3 A To determine whether DprA and its B. subtilis counterpart, Smf, require a free end for binding, we investigated their interaction with circular FX174 (FX) ssDNA. When increasing concentrations of the proteins were added to FX ssDNA, the mobility of ssDNA in native agarose gels decreased progressively This indicates that a free end on the substrate oligonucleotide is not necessary for Smf binding. smf-smf 1 12 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey physical association physical association S2 C Yeast two-hybrid assays revealing DprA and Smf self-interactions. q97qf0-q97qf0 1 9 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey physical association physical association S2 C Yeast two-hybrid assays revealing DprA and Smf self-interactions. reca-q97qf0 3 9 unspecified role unspecified role prey prey 5 unspecified role unspecified role bait bait c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association 5 A SDS-polyacrylamide gel electrophoresis of eluted proteins revealed two major bands (Figure 5A), with electrophoretic mobilities expected for SpRecA-His6 and DprA. Mass spectrometry of a trypsin digest of the purified two major bands unambiguously identified the proteins smf-reca 1 11 unspecified role unspecified role prey prey 12 unspecified role unspecified role bait bait physical association physical association 5 B in specific yeast two-hybrid assays. Homospecific interactions were detected between Smf and full-size BsRecA reca-atp 2 10 stimulator stimulator unspecified role unspecified role 7 enzyme target enzyme target substrate substrate unspecified role unspecified role 13 enzyme enzyme unspecified role unspecified role enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction We first monitored the effect of DprA on the ssDNA-dependent ATPase activity of EcRecA. We observed that at low concentration DprA, which itself exhibits no detectable ATPase activity (either in the presence or absence of ssDNA, data not shown), stimulates ssDNAdependent EcRecA ATPase S4 A The ATP used was labelled with 32P ([32P]gammaATP). SSB - SSB has a strong inhibitory effect on the ssDNA-dependent ATPase activity of EcRecA. DprA - DprA has a stimulatory effect on the ssDNA-dependent ATPase activity of EcRecA. smf-reca-1 1 11 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1549132]The shifted ranges (28-348) couldn't be applied as the new full feature sequence is different from the previous one. [EBI-1549132]28-347 [EBI-1549132]P16971,1 9 unspecified role unspecified role prey prey physical association physical association 5 B the AD fusion in which the first 27 residues of SpRecA are missing interacts with DprA.