Einzeltreffer — DigiBib

The mechanism through which the large serine recombinases bind DNA is poorly understood. Alignments of C31 integrase (Int) and its relatives indicate the presence of a conserved motif containing four cysteines resembling a zinc finger. Inductively coupled plasma-mass spectrometry (ICP-MS) confirmed that an Int monomer contains one atom of zinc. Pre-incubation of Int with ethylenediaminetetraacetic acid (EDTA) was detrimental for both recombination activity and DNA binding affinities but full activity could be restored by adding back Zn(2+). Mutations in the cysteines and other highly conserved residues yielded proteins that were hypersensitive to proteases, suggesting that without zinc the domain is unfolded. Substitutions in the highly charged region between the conserved cysteines led to lowered DNA binding affinities while circular dichroism revealed that these variant Ints were not greatly affected in overall folding. Int was protected from inhibition by EDTA when DNA containing an attachment site was present suggesting that the zinc finger and the DNA are in close proximity. A truncated mutant of Int, hInt V371S(UGA), lacking the putative zinc finger could bind DNA with low affinity. The data are consistent with there being at least two DNA binding motifs in Int one of which is the zinc finger-like motif.

Titel:	Zinc is essential for high-affinity DNA binding and recombinase activity of ΦC31 integrase.
Autor/in / Beteiligte Person:	McEwan, AR ; Raab, A ; Kelly, SM ; Feldmann, J ; Smith, MC
Zeitschrift:	Nucleic acids research, Jg. 39 (2011-08-01), Heft 14, S. 6137-47
Veröffentlichung:	1992- : Oxford : Oxford University Press ; <i>Original Publication</i>: London, Information Retrieval ltd., 2011
Medientyp:	academicJournal
ISSN:	1362-4962 (electronic)
DOI:	10.1093/nar/gkr220
Schlagwort:	Amino Acid Sequence Amino Acid Substitution Attachment Sites, Microbiological Chelating Agents pharmacology Circular Dichroism Cysteine chemistry DNA-Binding Proteins genetics DNA-Binding Proteins metabolism Edetic Acid pharmacology Integrases genetics Integrases metabolism Molecular Sequence Data Protein Binding Protein Stability Protein Structure, Secondary Recombination, Genetic Sequence Alignment Sequence Deletion Viral Proteins genetics Viral Proteins metabolism Zinc analysis Zinc metabolism Zinc Fingers DNA-Binding Proteins chemistry Integrases chemistry Siphoviridae enzymology Viral Proteins chemistry Zinc physiology
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article; Research Support, Non-U.S. Gov't Language: English [Nucleic Acids Res] 2011 Aug; Vol. 39 (14), pp. 6137-47. <i>Date of Electronic Publication: </i>2011 Apr 20. MeSH Terms: DNA-Binding Proteins / chemistry ; Integrases / chemistry ; Siphoviridae / enzymology ; Viral Proteins / chemistry ; Zinc / *physiology ; Amino Acid Sequence ; Amino Acid Substitution ; Attachment Sites, Microbiological ; Chelating Agents / pharmacology ; Circular Dichroism ; Cysteine / chemistry ; DNA-Binding Proteins / genetics ; DNA-Binding Proteins / metabolism ; Edetic Acid / pharmacology ; Integrases / genetics ; Integrases / metabolism ; Molecular Sequence Data ; Protein Binding ; Protein Stability ; Protein Structure, Secondary ; Recombination, Genetic ; Sequence Alignment ; Sequence Deletion ; Viral Proteins / genetics ; Viral Proteins / metabolism ; Zinc / analysis ; Zinc / metabolism ; Zinc Fingers References: Nucleic Acids Res. 2003 Jul 1;31(13):3503-6. (PMID: 12824354) ; Mol Microbiol. 2006 May;60(3):591-601. (PMID: 16629663) ; J Mol Biol. 2005 Jun 3;349(2):331-48. (PMID: 15890199) ; Biochem Soc Trans. 2010 Apr;38(2):388-94. (PMID: 20298189) ; Nucleic Acids Res. 2004 May 11;32(8):2607-17. (PMID: 15141031) ; Proc Natl Acad Sci U S A. 1998 May 12;95(10):5505-10. (PMID: 9576912) ; Cell. 1990 Dec 21;63(6):1323-9. (PMID: 2175678) ; Proc Natl Acad Sci U S A. 2005 May 10;102(19):6907-12. (PMID: 15863623) ; Nucleic Acids Res. 2009 Aug;37(14):4764-73. (PMID: 19515935) ; Mol Microbiol. 2000 Oct;38(2):232-41. (PMID: 11069650) ; Biopolymers. 2008 May;89(5):392-400. (PMID: 17896349) ; Nucleic Acids Res. 2007;35(10):3407-19. (PMID: 17478521) ; Mol Microbiol. 2002 Apr;44(2):299-307. (PMID: 11972771) ; Mol Microbiol. 2004 Mar;51(6):1719-28. (PMID: 15009897) ; Mol Cell. 2003 Nov;12(5):1101-11. (PMID: 14636570) ; Nucleic Acids Res. 2001 Jul 1;29(13):2860-74. (PMID: 11433033) ; Curr Gene Ther. 2006 Dec;6(6):633-45. (PMID: 17168696) ; Nucleic Acids Res. 2008 Jul;36(12):3879-91. (PMID: 18502775) ; Nucleic Acids Res. 2003 Jan 15;31(2):532-50. (PMID: 12527760) ; Trends Biochem Sci. 2007 Feb;32(2):63-70. (PMID: 17210253) ; Annu Rev Biochem. 2010;79:233-69. (PMID: 20334529) ; Nucleic Acids Res. 1992 Aug 25;20(16):4137-44. (PMID: 1508708) ; Annu Rev Biochem. 2006;75:567-605. (PMID: 16756503) ; Protein Expr Purif. 2009 Feb;63(2):102-11. (PMID: 18845260) ; J Mol Biol. 1991 Dec 20;222(4):897-908. (PMID: 1762157) ; J Bacteriol. 2002 Oct;184(20):5746-52. (PMID: 12270833) ; Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W668-73. (PMID: 15215473) ; Structure. 2008 Aug 6;16(8):1275-86. (PMID: 18682229) Grant Information: B19375 United Kingdom Biotechnology and Biological Sciences Research Council; BB/F011687/1 United Kingdom Biotechnology and Biological Sciences Research Council; BB/H001212/1 United Kingdom Biotechnology and Biological Sciences Research Council Substance Nomenclature: 0 (Chelating Agents) ; 0 (DNA-Binding Proteins) ; 0 (Viral Proteins) ; 9G34HU7RV0 (Edetic Acid) ; EC 2.7.7.- (Integrases) ; J41CSQ7QDS (Zinc) ; K848JZ4886 (Cysteine) Entry Date(s): Date Created: 20110422 Date Completed: 20111017 Latest Revision: 20211020 Update Code: 20240513 PubMed Central ID: PMC3152356

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Zinc is essential for high-affinity DNA binding and recombinase activity of ΦC31 integrase.