Celina Pinto/Sandbox 211: Difference between revisions
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The T5 5' exonuclease is 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. 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. | The T5 5'-exonuclease is 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. 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. | ||
During the DNA replication processes, the T5 5' exonuclease plays an important role by removing RNA primers from the 5’ end of the Okazaki fragment. This allows the ligation between the Okazaki fragment and the 3’ end of upstream DNA fragment. | During the DNA replication processes, the T5 5'-exonuclease plays an important role by removing RNA primers from the 5’ end of the Okazaki fragment. This allows the ligation between the Okazaki fragment and the 3’ end of upstream DNA fragment. | ||
Enzyme activity and DNA binding depends on the pH. At low pH, the association constant of the enzyme is maximal whereas the turnover number of the enzyme is maximal at high pH. It’s due to the protonation of Lys83, which is a catalytic residue in the active site. | Enzyme activity and DNA binding depends on the pH. At low pH, the association constant of the enzyme is maximal whereas the turnover number of the enzyme is maximal at high pH. It’s due to the protonation of Lys83, which is a catalytic residue in the active site. | ||
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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. | 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. | 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. | 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. | 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. | ||
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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. | 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. | 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. | 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 activity of the enzyme changes with the metal ion, which means that for example the T5 | The activity of the enzyme changes with the metal ion, which means that for example the T5 5’-exonuclease is more active with Mn2+ than Co2+ or Mg2+ because Mn2+ ions bind most strongly to the protein. | ||
The T5 5' exonuclease is inhibited in presence of Ca2+ ions. It is a competitive inhibition which means that the Ca2+ binds to the metal binding site and doesn't permit the binding of the catalytic divalent metal ion. | The T5 5'-exonuclease is inhibited in presence of Ca2+ ions. It is a competitive inhibition which means that the Ca2+ binds to the metal binding site and doesn't permit the binding of the catalytic divalent metal ion. | ||
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The T5 5’ exonuclease is used for plasmid and oligonucleotide mutagenesis. It can be used to destroy anything apart from pure closed circular plasmid, so it is useful to increase transfection efficiency and to reduce background in cloning experiments. T5 | The T5 5’-exonuclease is used for plasmid and oligonucleotide mutagenesis. It can be used to destroy anything apart from pure closed circular plasmid, so it is useful to increase transfection efficiency and to reduce background in cloning experiments. T5 5’-exonuclease is also used to remove denatured DNA from alkaline-based plasmid purification for improved cloning procedures. | ||
== '''References''' == | == '''References''' == | ||