Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1
Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of...
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| Veröffentlicht in: | Biochemistry (Moscow) 2020-05, Vol.85 (5), p.594-603 |
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| creator | Iakovlev, D. A. Alekseeva, I. V. Kuznetsov, N. A. Fedorova, O. S. |
| description | Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme’s tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1. |
| doi_str_mv | 10.1134/S0006297920050089 |
| format | Article |
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A. ; Alekseeva, I. V. ; Kuznetsov, N. A. ; Fedorova, O. S.</creator><creatorcontrib>Iakovlev, D. A. ; Alekseeva, I. V. ; Kuznetsov, N. A. ; Fedorova, O. S.</creatorcontrib><description>Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme’s tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.</description><identifier>ISSN: 0006-2979</identifier><identifier>EISSN: 1608-3040</identifier><identifier>DOI: 10.1134/S0006297920050089</identifier><identifier>PMID: 32571189</identifier><language>eng</language><publisher>Moscow: Pleiades Publishing</publisher><subject>2-Aminopurine ; Amino Acid Sequence ; Amino acids ; Analysis ; Arginine - chemistry ; Biochemistry ; Biomedical and Life Sciences ; Biomedicine ; Bioorganic Chemistry ; Catalysis ; Catalytic Domain ; Chemical compounds ; Deoxyribonucleic acid ; DNA ; DNA Damage ; DNA glycosylase ; DNA Repair ; Energy transfer ; Enzymes ; Ethylenediaminetetraacetic acid ; Fluorescence ; Fluorescence resonance energy transfer ; Fluorophores ; Histidine - chemistry ; Homology ; Humans ; Intermediates ; Kinetics ; Life Sciences ; Microbiology ; Molecular Dynamics Simulation ; Nucleotides ; Parameter identification ; Pyrimidines ; Reaction intermediates ; Reaction kinetics ; Recognition ; Residues ; Sequence Homology ; SMUG1 protein ; Substrate Specificity ; Substrates ; Tryptophan ; Uracil ; Uracil - metabolism ; Uracil-DNA glycosidase ; Uracil-DNA Glycosidase - chemistry ; Uracil-DNA Glycosidase - genetics ; Uracil-DNA Glycosidase - metabolism</subject><ispartof>Biochemistry (Moscow), 2020-05, Vol.85 (5), p.594-603</ispartof><rights>The Author(s) 2020</rights><rights>COPYRIGHT 2020 Springer</rights><rights>The Author(s) 2020.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c434t-ff886673f54681598640b8569c746fcc3a46d53e787f74c280daf4cf956b64d23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1134/S0006297920050089$$EPDF$$P50$$Gspringer$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1134/S0006297920050089$$EHTML$$P50$$Gspringer$$Hfree_for_read</linktohtml><link.rule.ids>314,776,780,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/32571189$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Iakovlev, D. A.</creatorcontrib><creatorcontrib>Alekseeva, I. V.</creatorcontrib><creatorcontrib>Kuznetsov, N. A.</creatorcontrib><creatorcontrib>Fedorova, O. S.</creatorcontrib><title>Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1</title><title>Biochemistry (Moscow)</title><addtitle>Biochemistry Moscow</addtitle><addtitle>Biochemistry (Mosc)</addtitle><description>Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme’s tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.</description><subject>2-Aminopurine</subject><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Analysis</subject><subject>Arginine - chemistry</subject><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>Bioorganic Chemistry</subject><subject>Catalysis</subject><subject>Catalytic Domain</subject><subject>Chemical compounds</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA Damage</subject><subject>DNA glycosylase</subject><subject>DNA Repair</subject><subject>Energy transfer</subject><subject>Enzymes</subject><subject>Ethylenediaminetetraacetic acid</subject><subject>Fluorescence</subject><subject>Fluorescence resonance energy transfer</subject><subject>Fluorophores</subject><subject>Histidine - chemistry</subject><subject>Homology</subject><subject>Humans</subject><subject>Intermediates</subject><subject>Kinetics</subject><subject>Life Sciences</subject><subject>Microbiology</subject><subject>Molecular Dynamics Simulation</subject><subject>Nucleotides</subject><subject>Parameter identification</subject><subject>Pyrimidines</subject><subject>Reaction intermediates</subject><subject>Reaction kinetics</subject><subject>Recognition</subject><subject>Residues</subject><subject>Sequence Homology</subject><subject>SMUG1 protein</subject><subject>Substrate Specificity</subject><subject>Substrates</subject><subject>Tryptophan</subject><subject>Uracil</subject><subject>Uracil - metabolism</subject><subject>Uracil-DNA glycosidase</subject><subject>Uracil-DNA Glycosidase - chemistry</subject><subject>Uracil-DNA Glycosidase - genetics</subject><subject>Uracil-DNA Glycosidase - metabolism</subject><issn>0006-2979</issn><issn>1608-3040</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>C6C</sourceid><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp1kctu1DAUhi0EotPCA7BBltiwSfE99nLUwgxSKVLLrCOPL8FVYhc7Wczb42haKm7ywjrnfP-vcwHgDUbnGFP24RYhJIhqFUGIIyTVM7DCAsmGIoaeg9VSbpb6CTgt5a6GBCn6EpxQwluMpVqBfJMGB5OH69wTRqGOFm5DIVTBG1eCnV2BIcLpu6uxSX0MU0hxEVzqUffOwuvZDC5NwVZyf4DbedQR7rI2YWgur9dwMxxMKodBFwdvv-w2-BV44fVQ3OuH_wzsPn38drFtrr5uPl-srxrDKJsa76UUoqWeMyExV1IwtJdcKNMy4Y2hmgnLqWtl61tmiERWe2a84mIvmCX0DLw_-t7n9KPOMXVjKMYNg44uzaUjDAsiqjmv6Ls_0Ls051i7qxRihEmO5RPV68F1Ifo01TEX024t6vJ4XS6u1Pk_qPqsG4NJ0flQ878J8FFgciolO9_d5zDqfOgw6pY7d3_duWrePjQ870dnfykeD1sBcgRKLcXe5aeJ_u_6Ex9CrCM</recordid><startdate>20200501</startdate><enddate>20200501</enddate><creator>Iakovlev, D. 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In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme’s tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.</abstract><cop>Moscow</cop><pub>Pleiades Publishing</pub><pmid>32571189</pmid><doi>10.1134/S0006297920050089</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 2-Aminopurine Amino Acid Sequence Amino acids Analysis Arginine - chemistry Biochemistry Biomedical and Life Sciences Biomedicine Bioorganic Chemistry Catalysis Catalytic Domain Chemical compounds Deoxyribonucleic acid DNA DNA Damage DNA glycosylase DNA Repair Energy transfer Enzymes Ethylenediaminetetraacetic acid Fluorescence Fluorescence resonance energy transfer Fluorophores Histidine - chemistry Homology Humans Intermediates Kinetics Life Sciences Microbiology Molecular Dynamics Simulation Nucleotides Parameter identification Pyrimidines Reaction intermediates Reaction kinetics Recognition Residues Sequence Homology SMUG1 protein Substrate Specificity Substrates Tryptophan Uracil Uracil - metabolism Uracil-DNA glycosidase Uracil-DNA Glycosidase - chemistry Uracil-DNA Glycosidase - genetics Uracil-DNA Glycosidase - metabolism |
| title | Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1 |
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