DNA PHOTOLYASE By: Kaleena Mormann. Function DNA repair enzymes They repair CPD lesions caused by UV...

DNA PHOTOLYASEBy: Kaleena Mormann

Function

• DNA repair enzymes• They repair CPD lesions caused by UV damage.• By catalyzing the cleavage of the cyclobutane ring

CPD lesion

Photolyases are found:

Cofactors

• Must have FADH

OR

Mechanism (E. coli)

Figure from Biochemistry, 33, 1994.1

Crystal structure (E. coli)

Red: Trp306 Green: MCH cofactor Blue: Beta sheets Purple: FAD cofactor Yellow: Alpha helices

Pdb:1dnp

Active site (E.coli)

CPD (red) is flipped out of the DNA helix (green) and into active site of photolyase (gray ribbons), where the catalytic flavin cofactor (yellow) is ready for action. The other cofactor is present (blue).

Pdb:1dnp

Hydropathy plot

Amino Acid Sequence

W306 in E.coli

Trp 306 → has functional role as electron donor

Trp 359 & Trp 382 → are structurally important, they are found between the donor and acceptor in the electron transfer process.

E-B-B-B-A-A-

Domain

Mutations• Researchers mutated W277, W306, W316, W359 and W382.

• They found that there was no affect on the activity in vivo and it was concluded that W316, W359 and W382 have more of a structural role. In contrast, W277 and W306 appeared to have mainly functional roles in the enzyme when mutated. They do not affect overproduction but interfere with either binding (W277) or photorepair in vitro (W306).

• Trp277 in E. coli photolyase was replaced the residues: Arg, Glu, Gln, His, and Phe by site-specific mutagenesis.

• Properties of the mutant proteins indicate that W277 is involved in binding to DNA but not in chromophore binding or catalysis.

• Results: • Photoreduction reaction can be inhibited• Decrease in electron coupling

Inhibition of mechanism in E. coli

• What would happen if DNA photolyase was inhibited:• Bacterial death• Super-mutation

NOT GOOD

References• Li, Y. F., Heelis, P. F., and Sancar, A. (1991) Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen

atom donor essential for Flavin radical photoreduction and DNA repair in vitro. Biochemistry. 30, 6322-6329.

• Li, Y. F., and Sancar, A. (1990) Active site of Escherichia coli DNA photolyase: mutations at Trp277 alter the selectivity of the enzyme without affecting the quantum yield of photorepair. Biochemistry. 29, 5698-5706.

• Kavakli, I. H., and Sancar, A. (2004) Analysis of the role of intraprotein electron transfer in photoreactivation by DNA photolyase in vivo. Biochemistry. 43, 15103-15110.

• Weber, S. (2005) Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase. Biochimica et Biophysica Act. 1701, 1-23.

• Maul, M. J., Barends, T. R. M., Glas, A. F., Cryle, M. J., Domratcheva, T., Schneider, S., Schlichting, I., and Carell, T. (2008) Crystal structure and mechanism of a DNA (6-4) photolyase. Angew.Chem.Int.Ed. 47, 10076-10080.

• Sancar, A. (1994) Structure and function of DNA photolyase. Biochemistry. 33, 2-9.

• Mees, A., Klar, T., Gnau, P., Hennecke, U., Eker, A. P. M., Carell, T. and Essen, L.O. (2004) Crystal Structure of photolyase bound to CPD-like DNA lesion after in situ repair. Science. 306, 1789-1793.

• Komori, H., Masui, R., Kuramitsu, S., Yokoyama, S., Shibata, T., Inour, Y., Miki, K. (2001) Crystal structure of thermostable DNA photolyase: pyrimidine-dimer recognition mechanism. PNAS. 98, 13560-13565.

• Cheung, M. S., Daizadeh, I., Stuchebrukov, A. A., and Heelis, P. F. (1999) Pathways of electron transfer in Escherichia coli DNA photolyase: Try306 to FADH. Biophy. J. 76, 1241-1249.