Purification and characterization of DNA photolyases

Members of the photolyase/cryptochrome family of blue-light photoreceptors are monomeric proteins of 50-70 kDa that contain two noncovalently bound chromophores/cofactors: either folate or deazaflavin, which act as a photoantenna, and a two electron-reduced FAD, which acts as a catalytic cofactor. DNA photolyases bind their substrates with high affinity and specificity and subsequently use blue light as a cosubstrate for the in situ conversion of ultraviolet-induced cyclobutane pyrimidine dimers and (6-4) photoproducts to canonical bases, thereby restoring the integrity of DNA. The determinants for binding, as well as the mechanism of the photolysis reaction, have been studied extensively using highly purified enzyme. In contrast, neither the substrate nor the reaction catalyzed by the closely related cryptochromes has been identified. This chapter describes methods used to purify DNA photolyases from a variety of organisms using an Escherichia coli overexpression system, as well as the properties of the purified enzymes and some of the assays commonly used to study DNA binding and repair by these enzymes in vitro.