https://scholars.lib.ntu.edu.tw/handle/123456789/618543
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.author | Chang, Hui-Lan | en_US |
dc.contributor.author | KANG-YI SU | en_US |
dc.contributor.author | Goodman, Steven D | en_US |
dc.contributor.author | WERN-CHERNG CHENG | en_US |
dc.contributor.author | LIANG-IN LIN | en_US |
dc.contributor.author | YA-CHIEN YANG | en_US |
dc.contributor.author | SUI-YUAN CHANG | en_US |
dc.contributor.author | WOEI-HORNG FANG | en_US |
dc.date.accessioned | 2022-09-06T08:27:03Z | - |
dc.date.available | 2022-09-06T08:27:03Z | - |
dc.date.issued | 2022 | - |
dc.identifier.issn | 1940-087X | - |
dc.identifier.uri | https://scholars.lib.ntu.edu.tw/handle/123456789/618543 | - |
dc.description.abstract | Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening. | en_US |
dc.language.iso | en | en_US |
dc.publisher | JOURNAL OF VISUALIZED EXPERIMENTS | en_US |
dc.relation.ispartof | Journal of visualized experiments : JoVE | en_US |
dc.subject | ESCHERICHIA-COLI; AMPLIFICATION STRATEGY; SINGLE-NUCLEOTIDE; EXCISION; REPAIR; BASE; SUBSTRATE; CELLS; DEAMINATION; GLYCOSIDASE | en_US |
dc.title | Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis | en_US |
dc.type | journal article | en |
dc.identifier.doi | 10.3791/63089 | - |
dc.identifier.pmid | 35532273 | - |
dc.identifier.scopus | 2-s2.0-85129547935 | - |
dc.identifier.isi | WOS:000836742900017 | - |
dc.identifier.url | https://scholars.lib.ntu.edu.tw/handle/123456789/611369 | - |
dc.relation.journalvolume | 2022 | en_US |
dc.relation.journalissue | 182 | en_US |
item.languageiso639-1 | en | - |
item.cerifentitytype | Publications | - |
item.fulltext | no fulltext | - |
item.openairecristype | http://purl.org/coar/resource_type/c_6501 | - |
item.openairetype | journal article | - |
item.grantfulltext | none | - |
crisitem.author.dept | Clinical Laboratory Sciences and Medical Biotechnology | - |
crisitem.author.dept | Laboratory Medicine-NTUH | - |
crisitem.author.dept | Laboratory Medicine | - |
crisitem.author.dept | Clinical Laboratory Sciences and Medical Biotechnology | - |
crisitem.author.dept | Clinical Laboratory Sciences and Medical Biotechnology | - |
crisitem.author.dept | Clinical Laboratory Sciences and Medical Biotechnology | - |
crisitem.author.dept | Laboratory Medicine-NTUH | - |
crisitem.author.dept | Clinical Laboratory Sciences and Medical Biotechnology | - |
crisitem.author.dept | Laboratory Medicine-NTUH | - |
crisitem.author.orcid | 0000-0002-6538-9526 | - |
crisitem.author.orcid | 0000-0002-5536-5942 | - |
crisitem.author.orcid | 0000-0002-3561-1093 | - |
crisitem.author.orcid | 0000-0002-9981-210X | - |
crisitem.author.orcid | 0000-0002-6009-6724 | - |
crisitem.author.orcid | 0000-0003-4728-5931 | - |
crisitem.author.parentorg | College of Medicine | - |
crisitem.author.parentorg | National Taiwan University Hospital | - |
crisitem.author.parentorg | College of Medicine | - |
crisitem.author.parentorg | College of Medicine | - |
crisitem.author.parentorg | College of Medicine | - |
crisitem.author.parentorg | College of Medicine | - |
crisitem.author.parentorg | National Taiwan University Hospital | - |
crisitem.author.parentorg | College of Medicine | - |
crisitem.author.parentorg | National Taiwan University Hospital | - |
顯示於: | 醫學檢驗暨生物技術學系 |
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