Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease
Two approaches were used to discern critical amino acid residues for the function of the I-CeuI homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I-...
Saved in:
Published in | Nucleic acids research Vol. 25; no. 13; pp. 2610 - 2619 |
---|---|
Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
01.07.1997
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
Abstract | Two approaches were used to discern critical amino acid residues for the function of the I-CeuI homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I-CeuI is lethal in Escherichia coli, enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis. |
---|---|
AbstractList | Two approaches were used to discern critical amino acid residues for the function of the I-CeuI homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I-CeuI is lethal in Escherichia coli, enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The K sub(m) and k sub(cat) values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis. Two approaches were used to discern critical amino acid residues for the function of the I- Ceu I homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I- Ceu I is lethal in Escherichia coli , enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis. Two approaches were used to discern critical amino acid residues for the function of the I-CeuI homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I-CeuI is lethal in Escherichia coli, enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis. |
Author | Otis, Christian Lemieux, Claude Turmel, Monique Côté, Vincent |
AuthorAffiliation | Program in Evolutionary Biology, Canadian Institute for Advanced Research, Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Québec G1K 7P4, Canada. mturmel@rsvs.ulaval.ca |
AuthorAffiliation_xml | – name: Program in Evolutionary Biology, Canadian Institute for Advanced Research, Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Québec G1K 7P4, Canada. mturmel@rsvs.ulaval.ca |
Author_xml | – sequence: 1 givenname: Monique surname: Turmel fullname: Turmel, Monique email: mturmel@rsvs.ulaval.ca organization: Program in Evolutionary Biology, Canadian Institute for Advanced Research, Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Québec G1K 7P4, Canada – sequence: 2 givenname: Christian surname: Otis fullname: Otis, Christian organization: Program in Evolutionary Biology, Canadian Institute for Advanced Research, Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Québec G1K 7P4, Canada – sequence: 3 givenname: Vincent surname: Côté fullname: Côté, Vincent organization: Program in Evolutionary Biology, Canadian Institute for Advanced Research, Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Québec G1K 7P4, Canada – sequence: 4 givenname: Claude surname: Lemieux fullname: Lemieux, Claude organization: Program in Evolutionary Biology, Canadian Institute for Advanced Research, Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Québec G1K 7P4, Canada |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/9185572$$D View this record in MEDLINE/PubMed |
BookMark | eNqFkb9vEzEUxy1UVNLAyobkie1S-_x7YEBRSyJVYgGBOmD5bF9juLODfRfR_x6XRBFMSJY8fD7v-T1_r8BFTNED8BqjFUaKXEeTr1u2wmTVcoyegQUmvG2o4u0FWCCCWIMRlS_AVSnfEcIUM3oJLhWWjIl2Ab7dHNIwTyHVPmF4hDbF4vPBO2iig_0c7R82VBTGfcqTiRPMvgQ3-wJDhNPOw22z9vMW7tIY4gP00aU428Gb4l-C570Zin91upfg8-3Np_Wmufv4Ybt-f9dYItnUCMZbKRTpbd-7XhIjZUc7ZZwjQrQIc0NdT1Anse2wrbY0XCHJO8QE65AlS_Du2Hc_d6N31scpm0HvcxhNftTJBP0viWGnH9JBY8pFfXgJ3p7qc_pZN5v0GIr1w2CiT3PRQiHGST3_EzFHjBKFq7g6ijanUrLvz8NgpJ-S0_XHdcs0JvopuVrw5u8VzvopqsqbIw9l8r_O2OQfmgsimN58vddUUXEvv2y0JL8BVMun-A |
CitedBy_id | crossref_primary_10_1016_j_jmb_2022_167550 crossref_primary_10_1371_journal_pone_0035647 crossref_primary_10_1007_s11103_006_9009_y crossref_primary_10_1007_s40778_017_0077_5 crossref_primary_10_1139_b06_117 crossref_primary_10_1101_gad_1109003 crossref_primary_10_1016_S1097_2765_00_80146_X crossref_primary_10_1016_j_protis_2011_11_004 crossref_primary_10_1016_j_funbio_2013_10_002 crossref_primary_10_1016_S0022_2836_02_00912_9 crossref_primary_10_1128_JB_181_16_4734_4740_1999 crossref_primary_10_1007_s00018_009_0188_y crossref_primary_10_1093_genetics_166_2_721 crossref_primary_10_1111_j_1742_4658_2005_04669_x crossref_primary_10_1186_1471_2148_6_37 crossref_primary_10_1016_S0022_2836_03_00426_1 crossref_primary_10_1016_j_jmb_2008_07_010 crossref_primary_10_1146_annurev_micro_56_012302_160741 crossref_primary_10_1016_j_str_2006_03_009 crossref_primary_10_1073_pnas_1434268100 crossref_primary_10_1016_j_jviromet_2006_10_016 crossref_primary_10_1074_jbc_273_46_30524 crossref_primary_10_1074_jbc_274_15_10235 crossref_primary_10_1534_genetics_166_2_721 |
ContentType | Journal Article |
DBID | BSCLL CGR CUY CVF ECM EIF NPM AAYXX CITATION 7TM 7X8 5PM |
DOI | 10.1093/nar/25.13.2610 |
DatabaseName | Istex Medline MEDLINE MEDLINE (Ovid) MEDLINE MEDLINE PubMed CrossRef Nucleic Acids Abstracts MEDLINE - Academic PubMed Central (Full Participant titles) |
DatabaseTitle | MEDLINE Medline Complete MEDLINE with Full Text PubMed MEDLINE (Ovid) CrossRef Nucleic Acids Abstracts MEDLINE - Academic |
DatabaseTitleList | Nucleic Acids Abstracts MEDLINE - Academic MEDLINE |
Database_xml | – sequence: 1 dbid: NPM name: PubMed url: https://proxy.k.utb.cz/login?url=http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed sourceTypes: Index Database – sequence: 2 dbid: EIF name: MEDLINE url: https://proxy.k.utb.cz/login?url=https://www.webofscience.com/wos/medline/basic-search sourceTypes: Index Database |
DeliveryMethod | fulltext_linktorsrc |
Discipline | Anatomy & Physiology Chemistry |
EISSN | 1362-4962 |
EndPage | 2619 |
ExternalDocumentID | 10_1093_nar_25_13_2610 9185572 ark_67375_HXZ_4947Z8WH_8 |
Genre | Research Support, Non-U.S. Gov't Journal Article Comparative Study |
GroupedDBID | --- -DZ -~X .55 .GJ .I3 123 18M 1TH 29N 2WC 3O- 4.4 482 53G 5VS 5WA 6.Y 70E 85S A8Z AABMN AAFWJ AAMVS AAOGV AAPPN AAPXW AAUQX AAVAP AAWDT AAYJJ ABPTD ABQLI ABQTQ ABSAR ABSMQ ABXVV ACFRR ACGFO ACGFS ACIPB ACIWK ACMRT ACNCT ACPQN ACPRK ACUTJ ADBBV ADHZD AEGXH AEKPW AENEX AENZO AFFNX AFPKN AFRAH AFULF AFYAG AGKRT AHMBA AIAGR ALMA_UNASSIGNED_HOLDINGS ALUQC ANFBD AOIJS AQDSO AQVPL ASAOO ASPBG ATDFG ATTQO AVWKF AZFZN BAWUL BAYMD BCNDV BEYMZ BSCLL BTTYL C1A CAG CIDKT COF CS3 CXTWN CZ4 D0S DFGAJ DIK DPORF DPPUQ DU5 D~K E3Z EBD EBS EJD ELUNK EMOBN ESTFP F20 F5P FEDTE GROUPED_DOAJ GX1 H13 HH5 HVGLF HYE HZ~ H~9 IH2 KAQDR KC5 KQ8 KSI M49 MBTAY MVM M~E NTWIH NU- OAWHX OBC OBS OEB OES OJQWA OJZSN OVD O~Y P2P PB- PEELM PQQKQ QBD R44 RD5 RNI RNS ROL ROX ROZ RPM RXO RZF RZO SJN SV3 TCN TEORI TN5 TOX TR2 UHB WG7 WOQ X7H X7M XFK XSB XSW YSK ZA5 ZKX ZXP ~91 ~D7 ~KM 0R~ AAHBH CGR CUY CVF ECM EIF NPM AAYXX CITATION 7TM 7X8 5PM |
ID | FETCH-LOGICAL-c385t-75628793fcffdf83a88b4b9add3772016a4df30b81cb1c7568a69086b0575b0c3 |
IEDL.DBID | RPM |
ISSN | 0305-1048 1362-4962 |
IngestDate | Tue Sep 17 21:19:08 EDT 2024 Fri Aug 16 06:08:09 EDT 2024 Fri Aug 16 01:13:43 EDT 2024 Fri Aug 23 01:51:17 EDT 2024 Sat Sep 28 08:34:46 EDT 2024 Sun Mar 31 11:37:37 EDT 2024 |
IsDoiOpenAccess | false |
IsOpenAccess | true |
IsPeerReviewed | true |
IsScholarly | true |
Issue | 13 |
Language | English |
LinkModel | DirectLink |
MergedId | FETCHMERGED-LOGICAL-c385t-75628793fcffdf83a88b4b9add3772016a4df30b81cb1c7568a69086b0575b0c3 |
Notes | ark:/67375/HXZ-4947Z8WH-8 istex:0326DD43A76104D97CAEA3AE43970C609C4D1068 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
OpenAccessLink | https://academic.oup.com/nar/article-pdf/25/13/2610/6963477/25-13-2610.pdf |
PMID | 9185572 |
PQID | 16054391 |
PQPubID | 23462 |
PageCount | 10 |
ParticipantIDs | pubmedcentral_primary_oai_pubmedcentral_nih_gov_146779 proquest_miscellaneous_79056356 proquest_miscellaneous_16054391 crossref_primary_10_1093_nar_25_13_2610 pubmed_primary_9185572 istex_primary_ark_67375_HXZ_4947Z8WH_8 |
PublicationCentury | 1900 |
PublicationDate | 1997-07 1997-Jul-01 1997-7-1 19970701 |
PublicationDateYYYYMMDD | 1997-07-01 |
PublicationDate_xml | – month: 07 year: 1997 text: 1997-07 |
PublicationDecade | 1990 |
PublicationPlace | England |
PublicationPlace_xml | – name: England |
PublicationTitle | Nucleic acids research |
PublicationTitleAlternate | Nucleic Acids Research |
PublicationYear | 1997 |
Publisher | Oxford University Press |
Publisher_xml | – name: Oxford University Press |
SSID | ssj0014154 |
Score | 1.7622523 |
Snippet | Two approaches were used to discern critical amino acid residues for the function of the I-CeuI homing endonuclease: sequence comparison of subfamilies of... Two approaches were used to discern critical amino acid residues for the function of the I- Ceu I homing endonuclease: sequence comparison of subfamilies of... |
SourceID | pubmedcentral proquest crossref pubmed istex |
SourceType | Open Access Repository Aggregation Database Index Database Publisher |
StartPage | 2610 |
SubjectTerms | Amino Acid Sequence Base Sequence Chlorophyta - enzymology Chloroplasts - enzymology Conserved Sequence DNA - metabolism Endodeoxyribonucleases - chemistry Endodeoxyribonucleases - genetics Endodeoxyribonucleases - metabolism Escherichia coli Escherichia coli - enzymology Escherichia coli - genetics Evolution, Molecular Introns Kinetics Molecular Sequence Data Mutation Sequence Alignment Structure-Activity Relationship Substrate Specificity |
Title | Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease |
URI | https://api.istex.fr/ark:/67375/HXZ-4947Z8WH-8/fulltext.pdf https://www.ncbi.nlm.nih.gov/pubmed/9185572 https://search.proquest.com/docview/16054391 https://search.proquest.com/docview/79056356 https://pubmed.ncbi.nlm.nih.gov/PMC146779 |
Volume | 25 |
hasFullText | 1 |
inHoldings | 1 |
isFullTextHit | |
isPrint | |
link | http://utb.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwnV1Nb9QwEB3RIkQvCFoqQvnwAZVTNnHsrJ1jtWq1RSpwaMWqByzbcdSojbfa7lbtv2ecjxWL4MIlFzuK4xl73tjPzwCfdGqC6_C4cJzFnIssNibjsayYNdrhrGnCesfZ1_H0gn-Z5bP-UNhdT6v01tQjf9OMfH3VcitvG5sMPLHk-9kkjG5RJFuwJRgbMvR-5wADUicZ1SpscrkWamSJ14sky0eUjTBtSHfgWYHBKhfZRkh6Gnr34W9480_a5G9x6OQlvOgBJDnqGvoKnji_C3tHHpPn5pEckpbS2a6V78LzyXCd2x78PL7vvQyz45tHYgONenHvSqJ9SUJ461YFsahuWlDulwRz8brE5pHaE0SK5DSeuNUpuZo3GPGICzeBBD1kjISv4eLk-HwyjfvLFWLLZL6MBQIfiYOzslVVVpJpKQ03BU53DAE3AkHNywotJak11GJtqTGRlmMTAJ5JLduHbT_37g2QtOQVpRViBVbxMRfaCYSZBYIDQ9OyoBF8HvpX3XYaGqrb-2YK_1lluaJMBZtEcNh2_7qaXlwH5pnI1XR2qXjBxaX8MVUygo-DfRT2Y9jk0N7NV3eKYn4WThP_u0aQJAuqfBHsd_Zcf633hwjGG4Zelwcp7s0S9NBWkrvzyLf_--IB7HSyuIEG_A62l4uVe49gZ2k-tN6Nz_Nvs1-WawDc |
link.rule.ids | 230,315,733,786,790,891,27955,27956,53825,53827 |
linkProvider | National Library of Medicine |
linkToHtml | http://utb.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwnV1LT9wwEB61oAoufUAR6QsfKnpKNomdjXNEK1C2ZVEPrLriUCt2HBFBvGjJIuDXd5zHqovaQ3u2o2jsGc839ufPAJ8zX1rXYW6iGXUZi0NXypC5vKBKZhpXTWn3OyZnw3TKvs6iWXcp7LajVRolS89cV54pLxtu5U2lBj1PbPB9MrLRHSeD57CJ4RpGfY3enR1gSmpFoxqNTcZXUo10YLLFIIy8gHpYOPjb8CLBdBXF4VpS2rTje_8nxPmUOPlbJjp5BdPehpaAcuUta-mpxyfyjv9q5Gt42UFTctS2voFn2uzA7pHBsrx6IIekIYs2u_A7sDXqH4rbhZ_Hd53_Yt19_UCUJWgv7nROMpMTmzjb_UZsKqsG7puaYJVf5mg2KQ1BDErG7kgvx-RyXmEuJdq-MWKVljHHvoXpyfH5KHW7ZxtcRXlUuzFCKo5hX6iiyAtOM84lkwkupBShPELMjOUF-gAPlAwU9uYZluh8KC10lL6ie7Bh5kbvA_FzVgRBgSiEFmzI4kzHCGAThB0y8PMkcOBLP2_iplXnEO2pOhVoswgjEVBh59qBw2ZaV92yxZXltMWRSGcXgiUsvuA_UsEdOOjnXeA42uOTzOj58lYEWPnZe8p_72HFzqzenwN7rZ-s_tb5mQPDNQdatVuR7_UWdItG7Lt1g3f_--EBbKXnk1NxOj779h62W_FdSzb-ABv1Yqk_IqSq5acmgn4BV28hhw |
linkToPdf | http://utb.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwnV1LT9wwEB5RUFsufUBRQx_4UNFTnnY2zhFtWe22BXEo6pZDrTixRQTxrrZZBP31Heex6qL2wtmOIntmPN_Ynz8DfMgCaV2Huali1GUsiVwpI-ZyTXOZKVw1pd3vODkdjM_Z52k83QDe34VpSPu5LD1zXXmmvGy4lfMq93uemH92MrTRnaT-vND-I9jCkI2Svk7vzg8wLbXCUY3OJuMruUbqm2zhR7EXUg-Lh2AbHqeYsuIkWktMW3aOb_-FOu-TJ__KRqPn8KMfR0tCufKWtfTy3_ckHh8y0BfwrIOo5Kjt8RI2lNmB3SOD5Xl1Rw5JQxptduN34OmwfzBuF34e33R-jPX39R3JLVF7caMKkpmC2ATa7jtiU1k1sN_UBKv9ssChk9IQxKJk4g7VckIuZxXmVKLsWyNWcRlz7Ss4Hx1_G47d7vkGN6c8rt0EoRXH8Ne51oXmNONcMpnigkoR0iPUzFih0Rd4mMswx948w1KdD6SFkDLI6R5smplRr4EEBdNhqBGNUM0GLMlUgkA2Rfghw6BIQwc-9rYT81alQ7Sn61TgmEUUi5AKa28HDhvTrrpliyvLbUtiMZ5eCJay5IJ_HwvuwEFve4HzaI9RMqNmy18ixArQ3lf-fw8remZ1_xzYa31l9bfO1xwYrDnRqt2Kfa-3oGs0ot-tK-w_9MMDeHL2aSS-Tk6_vIHtVoPXco7fwma9WKp3iKxq-b4Joj9Y0yQH |
openUrl | ctx_ver=Z39.88-2004&ctx_enc=info%3Aofi%2Fenc%3AUTF-8&rfr_id=info%3Asid%2Fsummon.serialssolutions.com&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Evolutionarily+conserved+and+functionally+important+residues+in+the+I-+CeuI+homing+endonuclease&rft.jtitle=Nucleic+acids+research&rft.au=Turmel%2C+M&rft.date=1997-07-01&rft.issn=1362-4962&rft.eissn=1362-4962&rft.volume=25&rft.issue=13&rft.spage=2610&rft.epage=2619&rft_id=info:doi/10.1093%2Fnar%2F25.13.2610&rft.externalDBID=n%2Fa&rft.externalDocID=10_1093_nar_25_13_2610 |
thumbnail_l | http://covers-cdn.summon.serialssolutions.com/index.aspx?isbn=/lc.gif&issn=0305-1048&client=summon |
thumbnail_m | http://covers-cdn.summon.serialssolutions.com/index.aspx?isbn=/mc.gif&issn=0305-1048&client=summon |
thumbnail_s | http://covers-cdn.summon.serialssolutions.com/index.aspx?isbn=/sc.gif&issn=0305-1048&client=summon |