T5 exonuclease

T5 exonuclease
	C -terminal domain, according to PDB 1UT5
other names	5'-3 'exonuclease, T5 flap endonuclease, Escherichia phage T5 exonuclease, T5 D15 gene product
	Existing structure data : PDB 1EXN , PDB 1J5F
Mass / length primary structure	291 amino acids , 33,448 Da
Identifier
External IDs	UniProt : P06229 ;
Enzyme classification
EC, category	3.1.11.-
	Orthologist (Escherichia Phage T5)
Entrez	2777611
UniProt	P06229 ;
PubMed search	2777611

The T5 exonuclease ( English T5 flap endonuclease ) is an enzyme from the group of nucleases ( exonucleases ) and is produced by the bacteriophage T5 .

properties

During the infection of Escherichia coli, the T5 exonuclease is used by the bacteriophage in the DNA replication of the viral genome . As an exonuclease, it catalyzes the hydrolysis of the terminal nucleotides of DNA double strands with blunt ends in the 5 '→ 3' direction with the release of nucleoside -5'-phosphate. In the case of 5 'bifurcated DNA, the T5 exonuclease cuts as an endonuclease at the junction between single-stranded and double-stranded DNA. The single-stranded DNA must have a free 5 'end for this. The T5 exonuclease binds DNA with a pseudo-Y structure with a dissociation constant of 5 nM ⁻¹ . Cofactors are three divalent magnesium ions , two of which are necessary for nuclease activity. The reaction also takes place with other divalent cations besides calcium ions . The pH optimum for the exonuclease is 9.3 and 5.5 for the endonuclease.

Applications

The T5 exonuclease is used in biochemistry, among other things, for Gibson assembly .

Web links

MeSH T5 exonuclease

Individual evidence

↑ SJ Garforth, TA Ceska, D. Suck, JR Sayers: Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3 'exonuclease suggests separate mechanisms of endo- and exonucleolytic cleavage. In: Proceedings of the National Academy of Sciences . Volume 96, Number 1, January 1999, pp. 38-43, PMID 9874768 , PMC 15089 (free full text).
↑ K. Syson, C. Tomlinson, BR Chapados, JR Sayers, JA Tainer, NH Williams, JA Grasby: Three metal ions participate in the reaction catalyzed by T5 flap endonuclease. In: The Journal of biological chemistry. Volume 283, Number 42, October 2008, pp. 28741-28746, doi : 10.1074 / jbc.M801264200 , PMID 18697748 , PMC 2568906 (free full text).
^ SJ Garforth, D. Patel, M. Feng, JR Sayers: Unusually wide co-factor tolerance in a metalloenzyme; divalent metal ions modulate endo-exonuclease activity in T5 exonuclease. In: Nucleic acids research. Volume 29, Number 13, July 2001, pp. 2772-2779, PMID 11433022 , PMC 55779 (free full text).
^ RM Benoit, C. Ostermeier, M. Geiser, JS Li, H. Widmer, M. Auer: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost. In: PloS one. Volume 11, number 4, 2016, p. E0153158, doi : 10.1371 / journal.pone.0153158 , PMID 27073895 , PMC 4830597 (free full text).
^ DG Gibson: Enzymatic assembly of overlapping DNA fragments. In: Methods in enzymology. Volume 498, 2011, pp. 349-361, doi : 10.1016 / B978-0-12-385120-8.00015-2 , PMID 21601685 .

[1] SJ Garforth, TA Ceska, D. Suck, JR Sayers: Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3 'exonuclease suggests separate mechanisms of endo- and exonucleolytic cleavage. In: Proceedings of the National Academy of Sciences . Volume 96, Number 1, January 1999, pp. 38-43, PMID 9874768 , PMC 15089 (free full text).

[2] K. Syson, C. Tomlinson, BR Chapados, JR Sayers, JA Tainer, NH Williams, JA Grasby: Three metal ions participate in the reaction catalyzed by T5 flap endonuclease. In: The Journal of biological chemistry. Volume 283, Number 42, October 2008, pp. 28741-28746, doi : 10.1074 / jbc.M801264200 , PMID 18697748 , PMC 2568906 (free full text).

[3] SJ Garforth, D. Patel, M. Feng, JR Sayers: Unusually wide co-factor tolerance in a metalloenzyme; divalent metal ions modulate endo-exonuclease activity in T5 exonuclease. In: Nucleic acids research. Volume 29, Number 13, July 2001, pp. 2772-2779, PMID 11433022 , PMC 55779 (free full text).

[4] RM Benoit, C. Ostermeier, M. Geiser, JS Li, H. Widmer, M. Auer: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost. In: PloS one. Volume 11, number 4, 2016, p. E0153158, doi : 10.1371 / journal.pone.0153158 , PMID 27073895 , PMC 4830597 (free full text).

[5] DG Gibson: Enzymatic assembly of overlapping DNA fragments. In: Methods in enzymology. Volume 498, 2011, pp. 349-361, doi : 10.1016 / B978-0-12-385120-8.00015-2 , PMID 21601685 .