Literature summary for 4.2.99.18 extracted from

Georgiadis, M.M.; Luo, M.; Gaur, R.K.; Delaplane, S.; Li, X.; Kelley, M.R.
Evolution of the redox function in mammalian apurinic/apyrimidinic endonuclease (2008), Mutat. Res., 643, 54-63.

Search for more literature for 4.2.99.18

Cloned(Commentary)

Cloned (Comment)	Organism
expression vectors encoding the full-length zApe and zApe (33-310) proteins in pET15b are constructed using standard subcloning techniques and verified by DNA sequencing. The zApe vectors are transformed into Rosetta (DE3) Escherichia coli, cultures are grown in LB media	Danio rerio
The wild-type and mutant hApe1 proteins are expressed as GST-fusions in Escherichia coli	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
crystals of zApe (74 mg/ml in 10 mM HEPES, pH 7.5 buffer) are grown by hanging drop vapor diffusion at 20°C using 200 mM ammonium acetate, 100 mM Bis-Tris pH 5.5, 7% PEG 3350, 2% glycerol and 0.8 mM lead acetate as the precipitating solution. The structure is solved at a resolution of 49.15-2.3 A	Danio rerio
mutant C65A hApe1 protein is diluted to 10 mg/ml in 10 mM HEPES pH 7.5 and then crystallized by hanging drop vapor diffusion using 10 mM MES pH 6.0, 7.5 mM Sm(OAc)3, 4% dioxane, 10-20% PEG 8000 as the precipitating solution at 20°C. The structure is solved at a resolution of 33.0-1.9 A	Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
C138A	stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active	Homo sapiens
C208A	stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active	Homo sapiens
C296A	stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active	Homo sapiens
C310A	stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active	Homo sapiens
C65A	site-directed mutagenesis of a pET28A vector encoding human Ape1 (40-318) is performed, mutant is redox inactive	Homo sapiens
C93A	stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active	Homo sapiens
C99A	stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active	Homo sapiens
T58C	Mutant enzyme is redox active. Site-directed mutagenesis is performed on the full-length zebrafish Ape pET15b vector using Stratagene Quikchange kit and verified by DNA sequencing. The T58C zApe vector is then transformed into Rosetta Escherichia coli (DE3). Stable ovarian cancer Skov-3X cells with the NFkappaB-Luc gene are cotransfected with plasmid pcDNA-mutant and a Renilla luciferase control reporter vector pRL-CMV using lipofectamine.	Danio rerio

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
DNA	Homo sapiens	-	fragments of DNA	-	?
DNA	Danio rerio	-	fragments of DNA	-	?
additional information	Homo sapiens	enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others	?	-	?
additional information	Danio rerio	enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Danio rerio	-	zebrafish	-
Homo sapiens	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
First step is a Ni-NTA affinity chromatography. Then the Ni-NTA fractions containing zApe are combined and loaded on a pre-equilibrated MonoQ column after dilution to a final NaCl concentration of 50 mM. The protein is eluted using a linear NaCl gradient, and fractions containing the zApe are combined, digested with thrombin, and then subjected again to a final MonoQ column. The purified proteins are concentrated to 50-75 mg/ml. The mutant T58C is purified by using an Q-sepharose column as ion exchanger.	Danio rerio
Purification is done using the standard glutathione affinity purification protocol. The C65A mutant is purified as follows: cell suspension is centrifuged, supernatant is loaded on a Ni-NTA column and the protein is eluted with a linear imidazole gradient. Fractions containing C65A hApe1 are further purified on an S-Sepharose column with a linear NaCl gradient.	Homo sapiens

Purification (Comment)

Organism

First step is a Ni-NTA affinity chromatography. Then the Ni-NTA fractions containing zApe are combined and loaded on a pre-equilibrated MonoQ column after dilution to a final NaCl concentration of 50 mM. The protein is eluted using a linear NaCl gradient, and fractions containing the zApe are combined, digested with thrombin, and then subjected again to a final MonoQ column. The purified proteins are concentrated to 50-75 mg/ml. The mutant T58C is purified by using an Q-sepharose column as ion exchanger.

Danio rerio

Purification is done using the standard glutathione affinity purification protocol. The C65A mutant is purified as follows: cell suspension is centrifuged, supernatant is loaded on a Ni-NTA column and the protein is eluted with a linear imidazole gradient. Fractions containing C65A hApe1 are further purified on an S-Sepharose column with a linear NaCl gradient.

Homo sapiens

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	Cell-based analysis of redox activity for hApe1 Cys-mutants using an in vitro EMSA redox assay in which reduction of the trancription factor AP-1 by hApe1 results in a shifted band. Only C65A hApe1 is found to be redox inactive.	Homo sapiens
additional information	-	T58C zApe has near wild-type human Ape1 redox activity in the transactivation assay	Danio rerio

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
DNA	-	Homo sapiens	fragments of DNA	-	?
DNA	-	Danio rerio	fragments of DNA	-	?
additional information	enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others	Homo sapiens	?	-	?
additional information	enzyme has an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others	Danio rerio	?	-	?

Subunits

Subunits	Comment	Organism
monomer	C65A hApe1 in solution, determined by size exclusion chromatography	Homo sapiens
monomer	zApe in solution, determined by gel filtration chromatography	Danio rerio

Synonyms

Synonyms	Comment	Organism
Ape	-	Homo sapiens
Ape	-	Danio rerio
apurinic/apyrimidinic endonuclease	-	Homo sapiens
apurinic/apyrimidinic endonuclease	-	Danio rerio
hAPE	-	Homo sapiens
hAPE1	-	Homo sapiens
zApe	-	Danio rerio