AID up-mutants isolated using a high-throughput screen highlight the. immunity/cancer balance limiting DNA deaminase activity
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- Dominick Shaw
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1 AID up-muns isoled usin hih-hrouhpu sreen hihlih he immuniy/ner blne limiin DNA deminse iviy Men Wn, Zizhen Yn, Crisin Rd nd Mihel S. Neuberer Medil Reserh Counil Lborory of Moleulr Bioloy, Hills Rod, Cmbride CB2 0QH, Unied Kindom Correspondene should be ddressed o Mihel Neuberer (msn@mrlmb.m..uk); Tel: ; Fx: Nure Sruurl & Moleulr Bioloy: doi: /nsmb.1623
![[S] (µm) 0.005 0.01 0.02 0.05 0.1 0.005 0.01 0.02 0.05 0.1 0.005 0.01 0.02 0.05 0.1 1 min 5 min 20 min WT AID Mu1.](/docs-images/82/85104172/images/2-0.jpg "1 Mu7.")
![3 1/[V] (fmol min 1 n 1 ) 1 300 250 WT 200 150 100 50](/docs-images/82/85104172/images/2-1.jpg "0-20 M7.3 M1.")
![1 0 20 40 60 80 100 120 1/[S] (µm) 1 Supplemenry Fiure 1](/docs-images/82/85104172/images/2-2.jpg "Kinei nlysis of humn GST-AID upmuns.")
2 [S] (µm) min 5 min 20 min WT AID Mu1.1 Mu7.3 1/[V] (fmol min 1 n 1 ) WT M7.3 M /[S] (µm) 1 Supplemenry Fiure 1 Kinei nlysis of humn GST-AID upmuns. Deminse iviy of GST-fusions of wild ype AID nd of upmuns Mu1.1 nd Mu7.3 ws ssyed on vrious onenrions of he sinle-srnded oliodeoxyribonuleoide subsre s indied nd s desribed under Mehods. Mihelis-Menen kinei nlysis ws performed usin Grphpd Prism sofwre. The dedued vlues of K M for he wild ype, Mu1.1 nd Mu7.3 fusion proeins were 80, 80 nd 100 nm respeively. Nure Sruurl & Moleulr Bioloy: doi: /nsmb.1623
3 S 1 W 1 M 1 R 1 E 2 F 2 E 6 Humn MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELL 60 Fuu ---MLLPRKKFIYHYKNVRWARGRHETYLCFVVKRRVGPDTLTFDFGHLRNRSGCHVELL 57 Humn FLRYISD-----WDLDPGR----CYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTA 111 Fuu FLRYLGALCPGLWGYGAAGEKRLSYSVTWFCSWSPCVNCSIQLCQFLNNTPNLRLRIFVS 117 Humn RLYFCE-DRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSV 170 Fuu RLYFCDLEDSLEREGLRMLTKAGVRISVMSYKDYFYCWQKFVDCKKSNFKAWEELHQNSV 177 Humn RLSRQLRRILLPLYEVDDLRDAFRTLGL 198 Fuu RLTRKLNRILQ-AWDLEDLRDALKLLGF 204 I 3 G 1 S 1 L Y 4 R 4 L 4 G 3 Y P 1 Q 1 M 1 P P 2 H R 1 L 1 L 1 S 1 G 1 T 1 I 1 K 1 E 2 D 1 G 1 A 2 STOP FRAMESHIFT Supplemenry Fiure 2 Comprison of humn nd fuu AID upmuions. Humn nd fuu AID primry sequenes re lined usin CluslW2 ( The humn AID upmuions re hihlihed in red nd orne s desribed in Fiure 3. The fuu AID upmuions re in reen, hvin been idenified eiher beuse hey onsiue he sole muion in fuu upmun or beuse he residue ws mued in muliple fuu upmuns. The nure of he subsiuions re indied in boxes bove or below he hihlihed residues s in Fiure 3. The zin-oordinion moifs (HVE, PCYDC) nd reions of suesed polynuleoide on (FCEDRKA) re hihlihed by blue box. Nure Sruurl & Moleulr Bioloy: doi: /nsmb.1623
4 GGGGCCGTCACTGATTGCCGTTTTCTCCCCTCTCTCCTCTCCCTCTCCAGGTTCCCTGGTGCAGGCAGCGCTGACTCAGCCGGCCTCGGTGTCAGCAAAT CDR 1 CCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTGGCAGCTATGCTGGAAGTTACTATTATGGCTGGTACCAGCAGAAGTCTCCTGGCAGTGCCCCTG CDR 2 TCACTGTGATCTATGACAACGACAAGAGACCCTCGGACATCCCTTCACGATTCTCCGGTTCCAAATCCGGCTCCACAGCCACATTAACCATCACTGGGGT CDR 3 CCGAGCCGATGACGAGGCTGTCTATTTCTGTGGGAGCTACGAAGACAACAGTGGTGCTGCATTTGGGGCCGGGACAACCCTGACCGTCCTAGGTGAGTCG CTGACCTCGTCTCGGTCTTTCTTCCCCCAT 420 Supplemenry Fiure 3 Disribuion of muions in IVλ sequenes deermined from AID-WT nd AID-Mu7.3 DT40 rnsfens. The onsensus IVλ sequene ws deermined from prenl AID / ψv / sim + DT40 ells. Complemenriy deerminin reion (CDR) sequenes re hihlihed wih box. Lower se leers bove nd below he onsensus sequene show he nuleoide subsiuions in IVλ sequenes from sim sored ells, wih hose from AID-WT rnsfens in red nd AID-Mu7.3 in blue. For boh AID-WT nd AID-Mu7.3 rnsfens, he Vλ sequenes presened re in eh se ompilion of sequenes obined from wo sepre pools of sim sored ells. Mued sequenes from individul pools of ells h onined idenil ses of muions were removed from he nlysis boh here nd in he Pie hr in Fi. 5b o void repe ounin of he sme muion. Nure Sruurl & Moleulr Bioloy: doi: /nsmb.1623
5 Veor AID-WT Mu 7.3 Mu 8 IM GFP b Crl WT Mu 7.3 mk-wt mk-mu IG % GFP % % % % Supplemenry Fiure 4 Flow yomery plos of he iviy of AID upmuns in nibody diversifiion. () Represenive plos nlysin he surfe IM loss of AID / ψv / sim + DT40 ells sbly rnsfeed wih onsrus o-expressin he indied AID muns oeher wih GFP. (b) Represenive plos showin swihin o IG1 by AID-defiien B ells h hve been rnsdued wih GFP-(WT/Mu7.3) nd ulured in LPS+IL4. mk indies mued Kozk sequene preedin he odin sequene of AID in he veor, so s o redue he exen of AID overexpression. Nure Sruurl & Moleulr Bioloy: doi: /nsmb.1623
6 Supplemenry Tble 1. Mmmlin A3 nd AID sequenes used o enere Fiure 7. Speies Common nme Proein Aession number/ensembl ID Homo spiens Humn A3A (A3Z1) NM_ Homo spiens Humn A3B (A3Z2-Z1) NM_ Homo spiens Humn A3C (A3Z2) NM_ Homo spiens Humn A3DE (A3Z2-Z2) NM_ Homo spiens Humn A3F (A3Z2-Z2) NM_ Homo spiens Humn A3G (A3Z2-Z1) NM_ Homo spiens Humn A3H (A3Z3) NM_ M mul Mque A3A (A3Z1) ENSMMUG M mul Mque A3B (Z2Z1) XM_ XM_ M mul Mque A3C (A3Z2) NM_ M mul Mque A3DE (A3Z2-Z2) XM_ M mul Mque A3F (A3Z2-Z2) NM_ M mul Mque A3G (A3Z2-Z1) XM_ M mul Mque A3H (A3Z3) XM_ Bos urus Cow A3Z1 EU Bos urus Cow A3Z2 EU Bos urus Cow A3Z3 EU Ovis ries Sheep A3Z1 EU Ovis ries Sheep A3Z2 EU Ovis ries Sheep A3Z3 EU Sus srof Pi A3Z2 EU Sus srof Pi A3Z3 EU Tyssu ju Pery A3Z2-Z3 EU Equus bllus Horse A3Z1 XM_ Equus bllus Horse A3Z2 XM_ Equus bllus Horse A3Z3 XM_ Felis us C A3Z2-Z3 EF Cnis lupus Do A3Z1 XM_ Cnis lupus Do A3Z2 AACN Cnis lupus Do A3Z3 XM_ Mus musulus Mouse A3Z2-Z3 NM_ Rus R A3Z2-Z3 NM_ norveius Homo spiens Humn AID NM_ M mul Mque AID XM_ Bos urus Cow AID NM_ Ovis ries Sheep AID EE Sus srof Pi AID BP Tyssu ju Pery AID EU Equus bllus Horse AID XM_ Felis us C AID ENSFCAG Cnis lupus Do AID NM_ Mus musulus Mouse AID NM_ Rus norveius R AID XM_ Mque A3A nd C AID use Ensembl IDs. Nure Sruurl & Moleulr Bioloy: doi: /nsmb.1623
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