Literature summary for 4.1.99.3 extracted from

Sancar, G.B.; Sancar, A.
Purification and characterization of DNA photolyases (2006), Methods Enzymol., 408, 121-156.

Cloned(Commentary)

Cloned (Comment)	Organism
cloned and overexpressed in Escherichia coli by using plasmid pMS969 (TetR AmpR Phr+), a derivative of ptac12 and propagation in any RecA- strain carrying Flac iQ. Optimal levels of expression are obtained using freshly transformed cells. Single colony is inoculated into 5 ml of LB and incubated at 37°C with shaking for 8 h. 1 ml of preculture is used to prepare an overnight culture consisting of 100 ml of LB. 10 ml of the overnight culture are added to 1 liter of LB and incubated with vigorous shaking at 37°C until the A600: 0.6-0.8 (3.5-4 h), at which time 2.0 ml of 0.5 M IPTG is added. Incubation is continued for another 4 h, at which point the photolyase typically constitutes 10-15% of total cellular protein	Escherichia coli
cloned and overexpressed in Escherichia coli CSR603 (recA1 uvrA6 phr1) containing FlaciQ using plasmid pCB1241 carrying the DNA photolyase gene under the control of the tac promoter on a TetR derivative of pUNC09. For overexpression, 40 ml LB containing 20 g/ml of tetracycline with 0.4 ml of the stock culture is inoculated and incubate at 37°C for 1216 h with shaking. Four 1 liter cultures with 10 ml of the overnight culture are inoculated and incubated until the culture reaches A600: 0.81.0. IPTG is added to a final concentration of 1 mM, incubation for further 1216 h.	Saccharomyces cerevisiae
phr gene is expressed under the control of a tac promoter on plasmid pUNC1993 in Escherichia coli strain CSR603 FlaciQ. Conditions are similar as expression for the enzyme derived from Escherichia coli. With the exception that induction is with 1 mM IPTG and postinduction incubation at 37°C continues for 10 h prior to harvesting the cells.	Aspergillus nidulans

Crystallization (Commentary)

Crystallization (Comment)	Organism
crystal structure of DNA photolyase from Escherichia coli is representative for the entire family. Enzyme is composed of two domains: an N-terminal alpha/beta domain (residues 1-131) and a C-terminal alpha-helical domain (residues 204-471). The two domains are connected by a long and structured interdomain loop. The photoantenna chromophore (MTHF) is located in a shallow groove between the two domains, while the FADH cofactor is deeply buried within the alpha-helical domain and is tightly bound by hydrogen bonds and ionic interactions with at least 14 amino acids. Distance between the photoantenna and the catalytic chromophore is 16.8 A center to center. A surface potential representation of the enzyme reveals the presence of a positively charged groove running nearly two-thirds of the length of the molecule and, lying in the approximate center of the groove, a hole leading to the flavin cofactor.	Escherichia coli

Crystallization (Comment)

Organism

crystal structure of DNA photolyase from Escherichia coli is representative for the entire family. Enzyme is composed of two domains: an N-terminal alpha/beta domain (residues 1-131) and a C-terminal alpha-helical domain (residues 204-471). The two domains are connected by a long and structured interdomain loop. The photoantenna chromophore (MTHF) is located in a shallow groove between the two domains, while the FADH cofactor is deeply buried within the alpha-helical domain and is tightly bound by hydrogen bonds and ionic interactions with at least 14 amino acids. Distance between the photoantenna and the catalytic chromophore is 16.8 A center to center. A surface potential representation of the enzyme reveals the presence of a positively charged groove running nearly two-thirds of the length of the molecule and, lying in the approximate center of the groove, a hole leading to the flavin cofactor.

Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Aspergillus nidulans	-	-	-
Escherichia coli	-	-	-
Saccharomyces cerevisiae	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
cells are purified by ammonium sulfate precipitation, blue sepharose chromatography, Bio Gel P-100 chromatography and hydroxylapatite chromatography. The described procedure typically yields 15-25 mg of above 98% pure photolyase from 5 liters of culture (850 mg total protein). Quality of the enzyme may be gauged initially by the color of the preparation. Preparations that appear deep blue are of good quality. Oxidation of the neutral blue flavin radical chromophore to FADox yields preparations displaying a green or yellow color depending on the extent of oxidation.	Escherichia coli
cells are purified by ammonium sulfate precipitation, phenyl sepharose chromatography, blue sepharose chromatography, DNA cellulose chromatography and DEAE cellulose/blue sepharose tandem chromatography. The described procedure typically yields 2-4 mg of above 98% pure photolyase from 4 liters of culture. Lower yield compared to the Escherichia coli enzyme is due to a lower level of expression and a lower yield during the purification procedure. Yeast enzyme purification necessitates a different strategy for purification. In particular, lysis of cells in the presence of (NH4)2SO4 followed by fractionation on phenyl Sepharose is necessary to inhibit and subsequently remove a protease that cleaves 2 kDa from the amino terminus of Phr1.	Saccharomyces cerevisiae
one day purification: following growth and collection of cells from 5 L of culture, cells are suspended in 25 ml (per 17.5 g of cells) of bufferA (50mM HEPES (pH 7.0), 100mM NaCl, 10% sucrose (w/v), 10 mM DTT), lysed by sonication, and centrifuged. Supernatant is applied to a Blue Sepharose CL-6B column. After washing with 510 column volumes, column is developed with a gradient of 0.12 M KCl in buffer B (50 mM HEPES (pH 7.0), 10% (v/v) glycerol, 10 mM DTT). Combined blue-colored fractions are precipitated with NH4SO4 (0.43 g/ml). Pellet is dissolved in buffer B containing 50 mM NaCl, and applied to a HiPrep 26/10 desalting column, and eluted with the same buffer. Combined blue-colored fractions are applied to a heparin Sepharose CL-6B column, washed with 5 column volumes, and eluted with a gradient of 0.1-1M KCl in buffer B. Fractions are pooled and concentrated by ultrafiltration using C-30 membranes	Escherichia coli
protein is purified by ammonium sulfate precipitation, blue sepharose chromatography, DNA cellulose chromatography and BioGel P-100 chromatography. The described procedure typically yields 5 mg of above 90% pure photolyase from 5 liters of culture. Full activity of the enzyme is achieved only by postpurification supplementation with deazaflavin, as Escherichia coli does not produce deazaflavin	Aspergillus nidulans
rapid purification method: sufficient quality for use as repair reagents, but not sufficient purity for some photophysical experiments. 5 L of cells are grown, induced with IPTG, collected, and lysed. Following collection of the NH4SO4 precipitate, proteins are suspended in 10 ml of equilibration buffer (50 mM Tris (pH 7.5), 100 mM KCl, 1 mM EDTA, 10% glycerol, 10 mM beta-ME). Mix is dialyzed for at least 4 h against 1 L of equilibration buffer. Clear dialysate is loaded onto a 50 ml Blue Sepharose column, washed with 300 ml of equilibration buffer and 300 ml of wash buffer. Elution by washing the column with high salt elution buffer (50 mM Tris (pH 7.5), 2 M KCl, 1 mM EDTA, 10% glycerol, 10 mM beta-ME). Fractions are pooled and dialyzed against 20-50 volumes of 33 mM potassium phosphate buffer (33 mM KH2PO4 (pH 6.8), 1 mM EDTA, 10% glycerol, 10 mM beta-ME). Dialysate is applied to a 10 ml hydroxylapatite column equilibrated with 33 mM potassium phosphate buffer minus glycerol. Column is washed with 75 ml of 33 mM potassium phosphate buffer and column developed with a 75 ml gradient of 33-330 mM potassium phosphate buffer, followed by a 20 ml wash with 330 mM potassium phosphate buffer. This procedure yields up to 10 mg from peak fractions that are 90% pure.	Escherichia coli

Storage Stability

Storage Stability	Organism
-70°C, glycerol to a final concentration of 50% (v/v) is added to the photolyase	Escherichia coli
glycerol to a final concentration of 50% (v/v) is added to the photolyase, storage at -70°C	Aspergillus nidulans

Synonyms

Synonyms	Comment	Organism
DNA photolyase	-	Escherichia coli
DNA photolyase	-	Saccharomyces cerevisiae
DNA photolyase	-	Aspergillus nidulans
PHR	-	Aspergillus nidulans
PHR1	-	Saccharomyces cerevisiae