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Literature summary for 2.7.7.7 extracted from

  • Zhang, H.; Eoff, R.L.; Kozekov, I.D.; Rizzo, C.J.; Egli, M.; Guengerich, F.P.
    Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translesion synthesis past bulky N2-alkylguanine adducts (2009), J. Biol. Chem., 284, 3563-3576.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Saccharolobus solfataricus

Crystallization (Commentary)

Crystallization (Comment) Organism
the enzyme is mixed with DNA (1:1.2 molar ratio) in 20 mM Tris-HCl buffer (pH 8.0, 25 °C) containing 60 mM NaCl, 4% glycerol (v/v), and 5 mM 2-mercaptoethanol and then placed on ice for 1 h prior to incubation with 5 mM MgCl2 and 1 mM dGTP. Crystals are grown using the sitting drop/vapor-diffusion method with the reservoir solution containing 20 mM Tris-HCl (pH 8.0 at 25 °C), 15% polyethylene glycol 3350 (w/v), 60 mM NaCl, 5 mM MgCl2, and 4% glycerol (v/v). Two crystal structures of Dpo4 with a template N2-NaphG (in a post-insertion register opposite a 3-terminal C in the primer) are solved. One shows N2-NaphG in a syn conformation, with the naphthyl group located between the template and the Dpo4 “little finger” domain. The Hoogsteen face is within hydrogen bonding distance of the N4 atoms of the cytosine opposite N2-NaphG and the cytosine at the 2 position. The second structure shows N2-NaphG in an anti conformation with the primer terminus largely disordered Saccharolobus solfataricus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetic parameters for one-base incorporation opposite G and N2-alkyl G adducts by Dpo4. Steady-state kinetic parameters for next base extension from G (or N2-alkyl G):C (or T) template primer termini by Dpo4 Saccharolobus solfataricus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
deoxynucleoside triphosphate + DNAn Saccharolobus solfataricus
-
diphosphate + DNAn+1
-
?
deoxynucleoside triphosphate + DNAn Saccharolobus solfataricus P2
-
diphosphate + DNAn+1
-
?

Organism

Organism UniProt Comment Textmining
Saccharolobus solfataricus Q97W02
-
-
Saccharolobus solfataricus P2 Q97W02
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
deoxynucleoside triphosphate + DNAn
-
Saccharolobus solfataricus diphosphate + DNAn+1
-
?
deoxynucleoside triphosphate + DNAn the enzyme shows a limited decrease in catalytic efficiency (kcat/Km) for insertion of dCTP opposite a series of N2-alkylguanine templates of increasing size from (methyl (Me) to (9-anthracenyl)-Me (Anth)). Fidelity is maintained with increasing size up to (2-naphthyl)-Me (Naph). The catalytic efficiency increases slightly going from the N2-NaphG to the N2-AnthG substrate, at the cost of fidelity. A set of oligonucleotides differing only in their N2-substitution at a single G site is used in this study with Dpo4 Saccharolobus solfataricus diphosphate + DNAn+1
-
?
deoxynucleoside triphosphate + DNAn
-
Saccharolobus solfataricus P2 diphosphate + DNAn+1
-
?
deoxynucleoside triphosphate + DNAn the enzyme shows a limited decrease in catalytic efficiency (kcat/Km) for insertion of dCTP opposite a series of N2-alkylguanine templates of increasing size from (methyl (Me) to (9-anthracenyl)-Me (Anth)). Fidelity is maintained with increasing size up to (2-naphthyl)-Me (Naph). The catalytic efficiency increases slightly going from the N2-NaphG to the N2-AnthG substrate, at the cost of fidelity. A set of oligonucleotides differing only in their N2-substitution at a single G site is used in this study with Dpo4 Saccharolobus solfataricus P2 diphosphate + DNAn+1
-
?

Synonyms

Synonyms Comment Organism
DNA polymerase Dpo4
-
Saccharolobus solfataricus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetic parameters for one-base incorporation opposite G and N2-alkyl G adducts by Dpo4. Steady-state kinetic parameters for next base extension from G (or N2-alkyl G):C (or T) template primer termini by Dpo4 Saccharolobus solfataricus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetic parameters for one-base incorporation opposite G and N2-alkyl G adducts by Dpo4. Steady-state kinetic parameters for next base extension from G (or N2-alkyl G):C (or T) template primer termini by Dpo4 Saccharolobus solfataricus