Use of a molecular beacon based fluorescent method for assaying uracil DNA glycosylase (Ung) activity and inhibitor screening.
Journal
Biochemistry and biophysics reports
ISSN: 2405-5808
Titre abrégé: Biochem Biophys Rep
Pays: Netherlands
ID NLM: 101660999
Informations de publication
Date de publication:
Jul 2021
Jul 2021
Historique:
received:
07
12
2020
revised:
05
02
2021
accepted:
05
02
2021
entrez:
5
3
2021
pubmed:
6
3
2021
medline:
6
3
2021
Statut:
epublish
Résumé
Uracil DNA glycosylases are an important class of enzymes that hydrolyze the N-glycosidic bond between the uracil base and the deoxyribose sugar to initiate uracil excision repair. Uracil may arise in DNA either because of its direct incorporation (against A in the template) or because of cytosine deamination. Mycobacteria with G, C rich genomes are inherently at high risk of cytosine deamination. Uracil DNA glycosylase activity is thus important for the survival of mycobacteria. A limitation in evaluating the druggability of this enzyme, however, is the absence of a rapid assay to evaluate catalytic activity that can be scaled for medium to high-throughput screening of inhibitors. Here we report a fluorescence-based method to assay uracil DNA glycosylase activity. A hairpin DNA oligomer with a fluorophore at its 5' end and a quencher at its 3' ends was designed incorporating five consecutive U:A base pairs immediately after the first base pair (5' C:G 3') at the top of the hairpin stem. Enzyme assays performed using this fluorescent substrate were seen to be highly sensitive thus enabling investigation of the real time kinetics of uracil excision. Here we present data that demonstrate the feasibility of using this assay to screen for inhibitors of
Identifiants
pubmed: 33665381
doi: 10.1016/j.bbrep.2021.100954
pii: S2405-5808(21)00048-0
pmc: PMC7900708
doi:
Types de publication
Journal Article
Langues
eng
Pagination
100954Informations de copyright
© 2021 Published by Elsevier B.V.
Déclaration de conflit d'intérêts
The authors declare no conflicts of interest.
Références
Biophys J. 1969 Nov;9(11):1281-311
pubmed: 4982056
Biotechniques. 2001 Nov;31(5):1106-16, 1118, 1120-1
pubmed: 11730017
Mol Cell Probes. 2011 Oct-Dec;25(5-6):219-21
pubmed: 21933706
Annu Rev Biochem. 1982;51:61-87
pubmed: 6287922
J Virol. 1980 Jan;33(1):535-8
pubmed: 6245250
PLoS One. 2015 Mar 26;10(3):e0119760
pubmed: 25811866
Structure. 1997 Dec 15;5(12):1543-50
pubmed: 9438868
Nucleic Acids Res. 1985 Jan 25;13(2):319-35
pubmed: 3889834
J Mol Biol. 2002 Aug 23;321(4):579-90
pubmed: 12206774
J Biol Chem. 2001 May 18;276(20):17324-31
pubmed: 11278852
Biochem Biophys Res Commun. 2004 Jun 18;319(1):240-6
pubmed: 15158468
J Biol Chem. 2006 Feb 24;281(8):4983-92
pubmed: 16306042
Nucleic Acids Res. 1981 Jun 11;9(11):2599-613
pubmed: 7279657
Protein Sci. 2014 Dec;23(12):1667-85
pubmed: 25252105
Genome Biol. 2000;1(4):RESEARCH0007
pubmed: 11178247
J Biol Chem. 1980 Mar 25;255(6):2293-300
pubmed: 6766936
Appl Biochem Biotechnol. 2013 Dec;171(8):2030-9
pubmed: 24018846
EMBO J. 1998 Sep 1;17(17):5214-26
pubmed: 9724657
CSH Protoc. 2006 Jun 01;2006(1):
pubmed: 22485377
Methods Enzymol. 1980;65(1):75-85
pubmed: 6154877
J Bacteriol. 1990 Jun;172(6):2979-85
pubmed: 2345131
Biochemistry. 1997 Jun 17;36(24):7557-66
pubmed: 9200707
Proc Natl Acad Sci U S A. 1974 Sep;71(9):3649-53
pubmed: 4610583
Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12989-94
pubmed: 14569030
Nucleic Acids Res. 2001 Sep 15;29(18):3857-63
pubmed: 11557818
Proteins. 1999 Apr 1;35(1):13-24
pubmed: 10090282
Methods Enzymol. 1980;65(1):499-560
pubmed: 6246368
EMBO J. 1996 Jul 1;15(13):3442-7
pubmed: 8670846
Mutat Res. 2010 Mar 1;685(1-2):11-20
pubmed: 19909758
Biochemistry. 1991 Apr 23;30(16):4055-61
pubmed: 2018771
Bioorg Med Chem Lett. 2008 Sep 15;18(18):4959-62
pubmed: 18752947
Nature. 1995 Feb 9;373(6514):487-93
pubmed: 7845459
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Dec 1;62(Pt 12):1231-4
pubmed: 17142904
Protein Expr Purif. 1998 Jul;13(2):155-62
pubmed: 9675057
Eur J Biochem. 1998 Sep 15;256(3):580-8
pubmed: 9780234
J Biol Chem. 1977 May 25;252(10):3286-94
pubmed: 324994
Cell. 1995 Mar 24;80(6):869-78
pubmed: 7697717
Anal Biochem. 1977 May 1;79(1-2):544-52
pubmed: 68686
J Bacteriol. 2010 Dec;192(24):6439-46
pubmed: 20971917
Biochemistry. 1988 Sep 6;27(18):6742-51
pubmed: 3196681
J Biol Chem. 1980 Nov 10;255(21):10354-8
pubmed: 6776115
Oncogene. 2002 Dec 16;21(58):8935-48
pubmed: 12483510
J Biol Chem. 1993 Dec 25;268(36):26879-85
pubmed: 8262921
Acta Crystallogr D Biol Crystallogr. 2014 Aug;70(Pt 8):2093-100
pubmed: 25084329
Molecules. 2020 Jun 20;25(12):
pubmed: 32575795
J Biol Chem. 2003 Jul 4;278(27):24350-8
pubmed: 12679366
Acta Crystallogr D Biol Crystallogr. 2015 Jul;71(Pt 7):1514-27
pubmed: 26143923
Eur J Med Chem. 2015 Jun 5;97:582-611
pubmed: 25306174
J Biol Chem. 1983 Feb 10;258(3):1603-9
pubmed: 6822524
Biotechniques. 2004 Feb;36(2):266-70, 272, 274-5
pubmed: 14989091
Tuberculosis (Edinb). 2011 May;91(3):212-8
pubmed: 21371942
EMBO J. 1996 Dec 2;15(23):6662-70
pubmed: 8978692
J Biol Chem. 1982 Feb 10;257(3):1208-14
pubmed: 7056715
Oncotarget. 2018 Aug 3;9(60):31719-31743
pubmed: 30167090
Antimicrob Agents Chemother. 2011 Mar;55(3):1088-96
pubmed: 21189350
Bioorg Med Chem. 2015 Nov 15;23(22):7226-33
pubmed: 26602084