Sand box 211: Difference between revisions

[[Image:activity.jpg | thumb | left | The different substrates of the T5 5'-exonuclease]]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 on the figure on the left), the pseudo-Y (B on the figure on the left) and the 5'-overhanging hairpin (C on the figure on the left) substrates.  

<Structure load='1EXN' size='350' frame='true' align='right' caption='T5 phage exonuclease (PDB code [[1exn]])' scene='Insert optional scene name here' />

The active site possesses 8 conserved <scene name='Sand_box_211/8_residus/4'>acidic residues</scene> (Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201, Asp204) which interact with divalent metal ions. <scene name='Sand_box_211/Try82/3'>Tyr82</scene> 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 <ref>PMID:12126622</ref>.   

<scene name='Sand_box_211/Lys83/3'>Lys83</scene> is positioned in the helical arch region close to metal site 1. It has an important binding role as well as a catalytic role <ref>PMID: 20698567</ref>. 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 <ref>PMID:10364212</ref> <ref>PMID:9889266</ref>.  

<scene name='Sand_box_211/Lys215arg216lys241/3'>Lys215, Arg216 and Lys241</scene> are important for binding to the 5' overhanging hairpin substrate <ref>PMID:12084915</ref>. Furthermore, residues <scene name='Sand_box_211/Lys215216/2'>Lys215 and Arg216</scene> form part of a helix–loop–helix feature. <scene name='Sand_box_211/Arg33/2'>Arg33</scene> binds to a phosphodiester residue in the 3'end of the cleavage site.

[[Image:mg.jpg | thumb | left | The metal ions' interaction]]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. Metal ions participate in the enzymatic catalysis of phosphodiester bond in several ways. For example, they can act as a nucleophile or a general base. They also participate in the catalysis of phosphate diester hydrolysis reactions by interacting with the oxygens which are not involved in the scissile phosphate (figure left).