Sand box 211
DescriptionDescription
The T5 5'-exonuclease, also called T5 5'-3' exonuclease, is a member of the family of flap endonucleases (FEN), also known as 5'-nucleases, and is composed of 291 amino acids. Flap endonucleases are present in almost all living organisms. They participate in DNA replication, by removing the Okazaki fragments, and repair processes. In addition, they are able to cleave branched DNA by catalyzing the exonucleolytic hydrolysis of the phosphodiester bonds present in the DNA. Furthermore, they have an endonucleolytic activity which consists in cleaving DNA flap structures. Both activities are structure-specific because they only take place in presence of double strand-single strand junctions in bifurcated nucleic acid substrates like the flap (A), the pseudo-Y (B) and the 5'-overhanging hairpin (C) substrates.
The T5 5'-exonuclease is found in bacteriophages T5 and is coded by the gene D15. It has a length of 290 amino acids. It is called metalloenzyme because it has binding sites for divalent metal ions without which the enzyme isn't able to cut DNA. However, it can bind to DNA without binding a divalent metal ion.
Flap endonuclease activityFlap endonuclease activity
Fens are able to cleave 5'flap or « pseudo Y » structures one nucleotide into the double-stranded region downstream of a single-stranded 5'arm. They have also an exonucleolytic activity on free 5' ends of single-stranded or double-stranded DNA depending on the divalent metal ions which are bound to the enzyme. They hydrolize phosphate diester linkages between nucleic acids which requires at least two divalent metal ions independently bound to the enzyme. The exonucleolytic and endonucleolytic hydrolyses cleave the 3'-oxygen phosphorus bond generating products terminating in a 3'-hydroxyl group and a 5'-phosphate monoester.
StructureStructure
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T5 5'-exonuclease is a homodimer composed of two identical chains, and . Both chains contain a hole, bound by a helical arch composed of two helices in which hydrophobic and positively charged residues are located. The helical arch is situated in front of the active site and only single-stranded DNA can pass through it. Since the enzyme is able to cleave double-stranded DNA, the helical arch may be able to change conformation in order to free the active site. The active site possesses 8 conserved acidic residues (Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201, Asp204) which interact with divalent metal ions. Tyr82 is also a conserved residue located in the active site, but it doesn't seem to have an important role since its mutation doesn't dramatically change the affinity to bind DNA. Six residues (Arg33, Lys83, Arg172, Lys 196, Lys215, Arg216 and Lys241) near the active site permit binding to branched DNA. Lys83 is positioned in the helical arch region close to metal site 1. It has an important binding role as well as a catalytic role. It was shown that DNA binding is pH dependent which means that the T5 5' exonuclease requires protonation of Lys83 to be able to bind to DNA. The mechanism of the Lys83 in the catalytic activity is still unknown, but it has been proposed that Lys83 acts as a general base/acid activating water to attack the scissile phosphodiester bond and protonating the leaving oxygen. Lys196 is positioned between two metal sites. Its mutation perturbs metal ion binding. Lys215, Arg216 and Lys241 are important for binding to the 5' overhanging hairpin substrate. Furthermore, residues Lys215 and Arg216 form part of a helix–loop–helix feature. Arg33 binds to a phosphodiester residue in the 3' end of the cleavage site.
The reaction only takes place if at least two divalent metal ions are bound to the enzyme. However, the DNA binding doesn't need the presence of metal ions. They only participate in the catalysis of phosphate diester hydrolysis reactions by interacting with the oxygens which are not involved in the scissile phosphate. Following divalent metal ions permit the reaction to take place : Mn2+, Mg2+, Co2+, Zn2+, Fe2+ and Cu2+. However, the reaction is the most efficient with Mn2+ and Mg2+ as cofactors. Furthermore, it has been shown that T5 5' exonuclease is able to cleave double-stranded closed-circular plasmids with an Mn2+ cofactor although this enzyme normally is only able to cleave single-stranded 5' ends. The two binding sites for metal ions are located near acidic residues ( Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201 and Asp204) which are responsible for binding them. However, previous studies have shown that the enzyme needs at least three metal ions for the reaction. As most of the T5 5' exonuclease x-ray structures in the absence of substrate show only two divalent metal ions bound, it implies that the third metal ion binds only in the presence of substrate, to stabilize the enzyme-DNA complex, and has less affinity for the free enzyme. However, the reaction also takes place, if there are only two metal ions present which confirms the two-metal-ion mechanism (figure) and that only two metal ions are needed for the catalytic reaction. The T5 5' exonuclease is inhibited in presence of Ca2+ ions, but it is still unknown if they bind to the divalent metal ion site or if the enzyme possesses an inhibitory binding site.
ApplicationsApplications
In molecular biology, the T5 5' exonuclease is above all used for mutagenesis in plasmids or oligonucleotides thanks to its exo- and endonucleolytic activities. It permits to mutate double-stranded DNA by deleting some nucleotides.
ReferencesReferences
1.↑ Ceska TA, Sayers JR, Stier G, Suck D. A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease. Nature. 1996 Jul 4;382(6586):90-3. PubMed: 8657312[1] DOI: 10.1038/382090a0[2]
2.↑ Ceska TA, Suck D, Sayers JR. Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage. Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):38-43. PubMed: 9874768[3]
3.↑ Mark R.Tock, Elaine Frary, Jon R.Sayers and Jane A.Grasby. Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonucleas. The EMBO Journal Vol. 22 No. 5 pp. 995±1004, 200. doi:10.1093/emboj/cdg098[4]