Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Saccharolobus solfataricus |
Crystallization (Comment) | Organism |
---|---|
crystallization of mutant enzymes R332A and R332E in complex with DNA and dGTP (R332E(8-oxoG:A), R332E(8-oxoG:C), R332A(8-oxoG:A) and R332A(8-oxoG:C)). The R332E(8-oxoG:C) structure is crystallized by the hanging drop vapor diffusion technique, using a mixture of 14% polyethylene glycol 4000 (w/v), 0.1 M calcium acetate, and 20 mM HEPES (pH 7.3) as reservoir | Saccharolobus solfataricus |
Protein Variants | Comment | Organism |
---|---|---|
R322H | the His332 mutant exhibits faster forward rate constants relative to wild-type Dpo4. The kpol values for the His332 mutant incorporation opposite G and 8-oxoG are 3.6- and 4.6fold faster than for wild-type Dpo4. The nucleotide binding affinity trend is opposite that of wild-type Dpo4, Glu332, and Leu332, with tighter dCTP binding during bypass of G. As in the case of Ala332, the kinetic analysis indicates that His332 inserts dCTP opposite G with slightly greater efficiency than opposite 8-oxoG | Saccharolobus solfataricus |
R322L | kpol (forward rate of polymerization) values for the Leu332 mutant incorporation opposite G and 8-oxoG are 4.1- and 1.9fold higher than those for wild-type Dpo4. The Leu332 mutant is about 2fold more efficient at incorporation of dCTP opposite 8-oxoG compared with G | Saccharolobus solfataricus |
R331A/R322A | mutant has lower forward rate constants relative to wild-type enzyme for both G and 8-oxoG. The double mutant-catalyzed insertion of dCTP opposite 8-oxoG is about 4fold higher than dCTP insertion opposite G. The measured binding affinity of dCTP is tighter than that of wild-type Dpo4 for unmodified DNA, but the binding affinity of dCTP opposite 8-oxoG is similar to that observed for wild-type enzyme. The catalytic efficiency for dCTP incorporation increases about 4fold for unmodified DNA and decreases about 2fold for 8-oxoG-modified DNA. The mutant fails to incorporate dATP opposite 8-oxoG in the presteady-state experiments. It inserts dCTP opposite 8-oxoG with an about 200fold greater efficiency than it does dATP and the steady-state efficiency of dATP incorporation is decreased about 27fold relative to the wild-type enzyme | Saccharolobus solfataricus |
R332A | mutant enzyme displays a higher affinity (lower KD,dCTP) for dCTP when bound to the unmodified DNA compared with the KD,dCTP measured for mutant-catalyzed incorporation of dCTP opposite 8-oxoG. Wild-type enzyme shows a greater affinity for dCTP opposite to 8-oxoG-modified DNA. The catalytic efficiency of the mutant is 23fold higher at incorporation of C opposite G and 1.2fold lower than wild-type enzyme in dCTP incorporation opposite 8-oxoG | Saccharolobus solfataricus |
R332E | mutant enzyme retains fidelity against bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) that is similar to wild enzyme. A crystal structure of the mutant and 8-oxoG:C pair reveals water-mediated hydrogen bonds between Glu332 and the O-8 atom of 8-oxoG. The kpol (forward rate of polymerization) value for dCTP incorporation opposite G is 4.8fold higher than for wild-type enzyme. The kpol (forward rates of polymerization) value for dCTP incorporation opposite 8-oxoG is 3.5fold higher than wild-type enzyme insertion of dCTP opposite 8-oxoG. The catalytic efficiency of the Glu332 mutant is 2.3fold greater than wild-type Dpo4 for dCTP incorporation opposite G but 1.7fold less efficient than wild-type Dpo4 for incorporation opposite 8-oxoG | Saccharolobus solfataricus |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Saccharolobus solfataricus | Q97W02 | - |
- |
Saccharolobus solfataricus P2 | Q97W02 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Saccharolobus solfataricus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
deoxynucleoside triphosphate + DNAn | - |
Saccharolobus solfataricus | diphosphate + DNAn+1 | - |
? | |
deoxynucleoside triphosphate + DNAn | - |
Saccharolobus solfataricus P2 | diphosphate + DNAn+1 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
DNA polymerase IV | - |
Saccharolobus solfataricus |
Dpo4 | - |
Saccharolobus solfataricus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | transient-state kinetic analysis of Dpo4 mutants and dATP Incorporation opposite 8-oxoG.Transient-state kinetic analysis of Dpo4 mutants and dCTP incorporation opposite 8-oxoG | Saccharolobus solfataricus |