Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering.
Amino Acid Motifs
Amino Acid Sequence
Circoviridae
/ enzymology
Conserved Sequence
Crystallography, X-Ray
DNA Helicases
/ chemistry
DNA, Single-Stranded
/ chemistry
Deoxyribonuclease I
/ chemistry
Gene Library
Models, Molecular
Molecular Docking Simulation
Molecular Sequence Data
Nucleic Acid Conformation
Plant Viruses
/ enzymology
Protein Binding
Protein Conformation
Protein Engineering
/ methods
Recombinant Fusion Proteins
/ chemistry
Replication Origin
Sequence Alignment
Sequence Homology, Amino Acid
Single-Strand Specific DNA and RNA Endonucleases
/ chemistry
Substrate Specificity
Trans-Activators
/ chemistry
Viral Proteins
/ chemistry
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
25 01 2021
25 01 2021
Historique:
accepted:
14
12
2020
revised:
08
12
2020
received:
05
10
2020
pubmed:
8
1
2021
medline:
4
2
2021
entrez:
7
1
2021
Statut:
ppublish
Résumé
Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.
Identifiants
pubmed: 33410911
pii: 6067397
doi: 10.1093/nar/gkaa1248
pmc: PMC7826260
doi:
Substances chimiques
DNA, Single-Stranded
0
Recombinant Fusion Proteins
0
Trans-Activators
0
Viral Proteins
0
replication initiator protein
0
Deoxyribonuclease I
EC 3.1.21.1
Single-Strand Specific DNA and RNA Endonucleases
EC 3.1.30.1
DNA Helicases
EC 3.6.4.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
1046-1064Subventions
Organisme : NIGMS NIH HHS
ID : T32 GM008347
Pays : United States
Organisme : NIAMS NIH HHS
ID : T32 AR007612
Pays : United States
Organisme : NIGMS NIH HHS
ID : P30 GM124165
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM119483
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM118047
Pays : United States
Informations de copyright
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.
Références
Nat Biotechnol. 2013 Sep;31(9):839-43
pubmed: 23934178
BMC Genomics. 2018 Jun 19;19(1):475
pubmed: 29914351
Nat Methods. 2006 Dec;3(12):995-1000
pubmed: 17072308
Biotechnol Lett. 2019 Mar;41(3):357-362
pubmed: 30603832
Structure. 2003 Nov;11(11):1310-1
pubmed: 14604517
J Am Chem Soc. 2017 May 24;139(20):7030-7035
pubmed: 28481515
Angew Chem Int Ed Engl. 2019 Mar 22;58(13):4144-4162
pubmed: 30153374
Front Plant Sci. 2020 Jul 23;11:1131
pubmed: 32849693
J Mol Biol. 2007 Mar 23;367(2):473-87
pubmed: 17275023
J Virol. 2011 Feb;85(3):1182-92
pubmed: 21084480
Nat Methods. 2014 Apr;11(4):429-35
pubmed: 24531420
Proc Natl Acad Sci U S A. 2013 Feb 19;110(8):2804-9
pubmed: 23359708
Biochem Pharmacol. 2006 Nov 30;72(11):1453-9
pubmed: 16764828
Nat Methods. 2013 May;10(5):403-6
pubmed: 23503053
Nucleic Acids Res. 2018 May 4;46(8):4152-4163
pubmed: 29635476
Chem Biol. 2008 Feb;15(2):128-36
pubmed: 18291317
Nat Struct Mol Biol. 2017 Feb;24(2):131-139
pubmed: 27991903
Elife. 2020 Feb 04;9:
pubmed: 32014111
Nucleic Acids Res. 1992 Jul 11;20(13):3279-85
pubmed: 1630899
Biotechnol Adv. 2015 Nov 1;33(6 Pt 2):1141-61
pubmed: 25708387
Trends Biotechnol. 2013 May;31(5):305-12
pubmed: 23545167
Nat Methods. 2017 Sep;14(9):865-868
pubmed: 28759029
Methods Mol Biol. 2014;1196:255-78
pubmed: 25151169
Adv Virus Res. 2019;103:71-133
pubmed: 30635078
ACS Chem Biol. 2008 Jun 20;3(6):373-82
pubmed: 18533659
J Clin Pathol. 2016 Apr;69(4):279-91
pubmed: 26644521
Nat Commun. 2019 Jul 31;10(1):3425
pubmed: 31366885
Nucleic Acids Res. 1993 Jun 11;21(11):2541-7
pubmed: 8332451
Cell. 2019 Jun 27;178(1):229-241.e16
pubmed: 31230717
Proc Natl Acad Sci U S A. 2020 Feb 11;117(6):2767-2769
pubmed: 31988118
Trends Plant Sci. 2006 May;11(5):209-12
pubmed: 16616578
Nat Biotechnol. 2003 Jan;21(1):86-9
pubmed: 12469133
Mol Cell. 2002 Aug;10(2):327-37
pubmed: 12191478
Nucleic Acids Res. 2020 Jan 8;48(D1):D277-D287
pubmed: 31612957
Trends Plant Sci. 2020 Sep;25(9):897-911
pubmed: 32371058
Mol Cell. 2004 Feb 13;13(3):403-14
pubmed: 14967147
Structure. 2005 Oct;13(10):1533-44
pubmed: 16216584
Commun Biol. 2018 May 31;1:54
pubmed: 30271937
Nucleic Acids Res. 2017 Jul 3;45(W1):W89-W97
pubmed: 28431131
Nat Protoc. 2017 Jun;12(6):1198-1228
pubmed: 28518172
Front Microbiol. 2017 Nov 30;8:2353
pubmed: 29250047
Nat Rev Microbiol. 2013 Aug;11(8):525-38
pubmed: 23832240
Nat Rev Microbiol. 2013 Nov;11(11):777-88
pubmed: 24100361
Angew Chem Int Ed Engl. 2016 Mar 18;55(13):4348-52
pubmed: 26915475
J Mol Biol. 2006 May 5;358(3):857-69
pubmed: 16540117
Plant Sci. 2020 Mar;292:110410
pubmed: 32005374
Nat Rev Genet. 2008 Apr;9(4):267-76
pubmed: 18319742
Bioconjug Chem. 2020 Apr 15;31(4):1093-1106
pubmed: 31809024
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
pubmed: 20383002
Proc Natl Acad Sci U S A. 2003 Sep 30;100(20):11243-8
pubmed: 14504391
Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10310-5
pubmed: 12130667
Proc Natl Acad Sci U S A. 2017 Aug 8;114(32):E6526-E6535
pubmed: 28739894
Commun Biol. 2020 Jan 23;3(1):44
pubmed: 31974493
Br J Obstet Gynaecol. 1998 Feb;105(2):174-8
pubmed: 9501782
Chem Commun (Camb). 2019 Oct 15;55(83):12428-12446
pubmed: 31576822
Small. 2019 Jun;15(26):e1804044
pubmed: 30645016
Viruses. 2019 Jul 18;11(7):
pubmed: 31323869
Acta Crystallogr D Struct Biol. 2019 Oct 1;75(Pt 10):861-877
pubmed: 31588918
Cell. 2018 Nov 1;175(4):1141-1155.e16
pubmed: 30343902
J Virol. 2018 Aug 29;92(18):
pubmed: 29976661
PLoS One. 2010 Jan 22;5(1):e8850
pubmed: 20107513
Nucleic Acids Res. 2013 Jun;41(11):e119
pubmed: 23605040
Prenat Diagn. 2004 Jul;24(7):513-8
pubmed: 15300741
Nanotechnology. 2020 Apr 3;31(25):255102
pubmed: 32176872
N Engl J Med. 2004 Feb 5;350(6):586-97
pubmed: 14762186
Annu Rev Phytopathol. 2018 Aug 25;56:637-677
pubmed: 30149794
Acta Crystallogr F Struct Biol Commun. 2019 Dec 1;75(Pt 12):744-749
pubmed: 31797816
Biochemistry. 2007 May 29;46(21):6201-12
pubmed: 17472345
Curr Opin Mol Ther. 2009 Aug;11(4):442-7
pubmed: 19649989
Mol Cell. 2008 Feb 15;29(3):302-12
pubmed: 18280236
Nucleic Acids Res. 2014;42(16):10632-43
pubmed: 25123661
Trends Biochem Sci. 1998 Nov;23(11):434-8
pubmed: 9852762
Nat Methods. 2011 Aug 07;8(9):765-70
pubmed: 21822273