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Literature summary extracted from

  • Schoenbrunner, N.J.; Fiss, E.H.; Budker, O.; Stoffel, S.; Sigua, C.L.; Gelfand, D.H.; Myers, T.W.
    Chimeric thermostable DNA polymerases with reverse transcriptase and attenuated 3-5 exonuclease activity (2006), Biochemistry, 45, 12786-12795.
    View publication on PubMed

Application

EC Number Application Comment Organism
2.7.7.7 diagnostics rational design approach to creating modulated proofreading DNA polymerases which can be utilized in a highly sensitive long RT/PCR amenable to the clinical diagnostic setting Thermus sp.
2.7.7.7 diagnostics rational design approach to creating modulated proofreading DNA polymerases which can be utilized in a highly sensitive long RT/PCR amenable to the clinical diagnostic setting Thermotoga maritima

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.7.49 chimeric DNA polymerase, termed CS5 pol, constructed from T. Z05 pol and Tma pol and containing the 5'-3' nuclease domain from Thermus sp. Z05 DNA polymerase (residues 1-291) and the 3'-5' exonuclease and polymerase domains from Thermotoga maritima DNA polymerase (residues 292-893). This chimera retains thermostable DNA polymerase activity, as well as proofreading activity. Using the CS5 chimera, a series of mutant proteins is constructed in which the amino acid side chains are mutated to modulate the 3'-5' exonuclease activity. The chimeric DNA polymerases are overexpressed under the control of the lambda PL promoter are expressed in Escherichia coli Thermus sp.
2.7.7.49 chimeric DNA polymerase, termed CS5 pol, constructed from T. Z05 pol and Tma pol and containing the 5'-3' nuclease domain from Thermus sp. Z05 DNA polymerase (residues 1-291) and the 3'-5' exonuclease and polymerase domains from Thermotoga maritima DNA polymerase (residues 292-893). This chimera retains thermostable DNA polymerase activity, as well as proofreading activity. Using the CS5 chimera, a series of mutant proteins is constructed in which the amino acid side chains are mutated to modulate the 3'-5' exonuclease activity. The chimeric DNA polymerases are overexpressed under the control of the lambda PL promoter are expressed in Escherichia coli Thermotoga maritima

Protein Variants

EC Number Protein Variants Comment Organism
2.7.7.7 additional information construction of a chimeric DNA polymerase derived from Thermus species Z05 and Thermotoga maritima DNA polymerases. These chimeric DNA polymerases are fashioned using structure-based tools to identify amino acid residues involved in the substrate-binding site of the exonuclease domain of a thermostable DNA polymerase. Mutation of some of these residues results in proteins in which DNA polymerase activity is unaffected, while proofreading activity ranges from 60% of the wild-type level to undetectable levels. Kinetic characterization of the exonuclease activity indicates that the mutations affects catalysis much more than binding. On the basis of their specificity constants (kcat/KM), the mutant enzymes have a 5-15-fold stronger preference for a double-stranded mismatched substrate over a single-stranded substrate than the wild-type DNA polymerase, a desirable attribute for RT/PCR Thermus sp.
2.7.7.7 additional information construction of a chimeric DNA polymerase derived from Thermus species Z05 and Thermotoga maritima DNA polymerases. These chimeric DNA polymerases are fashioned using structure-based tools to identify amino acid residues involved in the substrate-binding site of the exonuclease domain of a thermostable DNA polymerase. Mutation of some of these residues results in proteins in which DNA polymerase activity is unaffected, while proofreading activity ranges from 60% of the wild-type level to undetectable levels. Kinetic characterization of the exonuclease activity indicates that the mutations affects catalysis much more than binding. On the basis of their specificity constants (kcat/KM), the mutant enzymes have a 5-15-fold stronger preference for a double-stranded mismatched substrate over a single-stranded substrate than the wild-type DNA polymerase, a desirable attribute for RT/PCR Thermotoga maritima
2.7.7.49 additional information chimeric DNA polymerase, termed CS5 pol, constructed from T. Z05 pol and Tma pol and containing the 5'-3' nuclease domain from Thermus sp. Z05 DNA polymerase (residues 1-291) and the 3'-5' exonuclease and polymerase domains from Thermotoga maritima DNA polymerase (residues 292-893). This chimera retains thermostable DNA polymerase activity, as well as proofreading activity. Using the CS5 chimera, a series of mutant proteins is constructed in which the amino acid side chains are mutated to modulate the 3'-5'?exonuclease activity. A thermoactive and thermostable enzyme with reverse transcriptase and 3'-5'exonuclease activity is described Thermotoga maritima
2.7.7.49 additional information chimeric DNA polymerase, termed CS5 pol, constructed from T. Z05 pol and Tma pol and containing the 5'-3' nuclease domain from Thermussp. Z05 DNA polymerase (residues 1-291) and the 3'-5' exonuclease and polymerase domains from Thermotoga maritima DNA polymerase (residues 292-893). This chimera retains thermostable DNA polymerase activity, as well as proofreading activity. Using the CS5 chimera, a series of mutant proteins is constructed in which the amino acid side chains are mutated to modulate the 3'-5' exonuclease activity. A thermoactive and thermostable enzyme with reverse transcriptase and 3'-5'?exonuclease activity is described Thermus sp.

Organism

EC Number Organism UniProt Comment Textmining
2.7.7.7 Thermotoga maritima
-
-
-
2.7.7.7 Thermus sp.
-
-
-
2.7.7.7 Thermus sp. Z05
-
-
-
2.7.7.49 Thermotoga maritima
-
-
-
2.7.7.49 Thermus sp.
-
-
-
2.7.7.49 Thermus sp. Z05
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.7.49
-
 Thermus sp.
2.7.7.49
-
 Thermotoga maritima

Synonyms

EC Number Synonyms Comment Organism
2.7.7.49 CS5 pol chimeric DNA polymerase, termed CS5 pol, constructed from T. Z05 pol and Tma pol Thermus sp.
2.7.7.49 CS5 pol chimeric DNA polymerase, termed CS5 pol, constructed from T. Z05 pol and Tma pol Thermotoga maritima
2.7.7.49 T. Z05 pol
-
 Thermus sp.
2.7.7.49 T. Z05 pol
-
 Thermotoga maritima