6-4 Photolyase binding

Thymine dimers can be removed by photoreactivation (2). A specific photolyase binds at the defect and, when illuminated, cleaves the dimer to yield two single bases again. 

Cryptochromes in the human eye have a considerable sequence and structure homology with the photolyases, binding both methylene tetrahydrofolate and FAD. They have the same DNA binding pocket as photolyase, although they do not catalyze the reduction of DNA pyrimidine dimers. They are found in the nucleus of cells of the inner layer of the retina, behind the rods and cones involved in vision (Section 2.3.1), and absorb blue light, with maximum absorbance at 420 nm. 

Photolyase binds to PyrOPyr in DNA with a second-order rate constant (Aon 10 —lO sec ) consistent with target location by three-dimen-... 

The Drosophila and Xenopus (6-4) photolyases appear to bind DNA containing a (6-4) photoproduct by three-dimensional diffusion and to make contacts around the lesion quite similar to the contacts made by photolyase with DNA containing a cyclobutane pyrimidine dimer (Hitomi et al, 1997 Zhao et al, 1997). The (6-4) photolyase, like the cyclobutane photolyase, binds to its cognate lesion in ssDNA and dsDNA with essentially equal affinities (Zhao et al, 1997). When bound to a dsDNA substrate, the enzyme confers single-strandedness to a 4-bp region around the lesion, and the presence of a mismatch across the (6—4) photoproduct increases the affinity of the enzyme for the substrate (Zhao et al, 1997). These three features of binding, that is, binding to substrate in ssDNA with... 

Clearly, all indications are that (6—4) photolyase binds DNA and repairs its substrate by a mechanism quite similar to that of classical photolyase. However, there appears to be a fundamental difference in the photochemical reaction catalyzed by the two enzymes. The quantum yield of repair by excited singlet-state flavin by classical photolyase is near unity, whereas the quantum yield of repair by excited flavin in (6-4) photolyase is 0.05-0.10. Whether this low quantum yield of repair by (6—4) photolyase is a result of the low efficiency of formation of the oxetane intermediate thermally, low efficiency of electron transfer from the flavin to the photoproduct, or low efficiency splitting of the oxetane anion coupled with high rate of back electron transfer is not known at present. Furthermore, it was found that (6-4) photolyase can photorepair the Dewar valence isomer of the (6-4) photoproduct (Taylor, 1994) that cannot form an oxetane intermediate, casting some doubt about the basic premise of the retro [2+2] reaction. However, the Dewar isomer is repaired with 300-400 lower quantum yield than the (6-4) photoproduct, and it has been proposed (Zhao et ai, 1997) that the Dewar isomer may be repaired by the enzyme through a two-photon reaction in which the first photon converts the Dewar isomer to the Kekule form and a second electron transfer reaction initiated by the second photon promotes the retro [2+2] reaction. 

Flavins — Riboflavin is first of all essential as a vitamin for humans and animals. FAD and FMN are coenzymes for more than 150 enzymes. Most of them catalyze redox processes involving transfers of one or two electrons. In addition to these well known and documented functions, FAD is a co-factor of photolyases, enzymes that repair UV-induced lesions of DNA, acting as photoreactivating enzymes that use the blue light as an energy source to initiate the reaction. The active form of FAD in photolyases is their two-electron reduced form, and it is essential for binding to DNA and for catalysis. Photolyases contain a second co-factor, either 8-hydroxy-7,8-didemethyl-5-deazariboflavin or methenyltetrahydrofolate. ... 

Carell has recently presented the study of a flavin amino acid chimera to model riboflavin in DNA photolyases [68]. This amino acid LI (Fig. 20) was synthesized in an enantiopure fashion by building the alloxazine ring onto the epsilon amine of lysine. This coenzyme chimera was applied to the problem of repairing DNA damage caused by UV irradiation. LI was incorporated into an 21-residue peptide, P-1, possessing the sequence of the DNA-binding domain of the helix-loop-helix transcription factor MyoD. 

Belanich et al.63 reported the removal of endotoxins from protein mixtures. Endotoxins in a bacterial extract containing a protein photolyase was passed through a stack of 10 disk membranes (Q-type, Sartorius). LAL assay was used to monitor the endotoxin levels after each pass. There was over 5 log reduction in endotoxin content after three passes through the membranes. The protein content was reduced during this process, however, the enzyme s specific activity was increased 35-fold. This study also determined that the binding capacity of the membrane was greater than 2.25 million EU/cm2 of membrane area. 

Figure 23.1. Schematic illustration of direct reversal of a pyrimidine dimer by the enzyme DNA photolyase. The enzyme binds to the pyrimidine dimer present in DNA. The square and triangle represent the two noncovalently bound chromophores that are present in all photolyases. The chromophores harness the energy of photoreactivating blue wavelengths of light and use them to catalyze the breakage of the pyrimidine dimer back to adjacent monomers. [Adapted fromFriedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A., and Ellenberger, T. (Eds.). DNA Repair and Mutagenesis, 2nd ed., ASM, Washington, D.C., 2006.]...

Figure 1 Reaction mechanism of DNA photolyases (A) mechanism of cyclobutane photolyase and (B) mechanism of (6-4) photolyase. Both photolyases harness blue light energy to remove UV-induced damage and contain two noncovalently bound chromophores. They bind UV-damaged DNA in a reaction that is light independent and carry out catalysis in a light-initiated cyclic electron transfer. In (6-4) photolyase, the (6-4) photoproduct is converted to a four-membered oxetane ring thermally (kT) before the photochemical reaction.

An example of direct repair is the photochemical cleavage of pyrimidine dimers. Nearly all cells contain a photoreactivating enzyme called DNA photolyase. The E. coli enzyme, a 35-kd protein that contains bound N lO-methenyltetrahydrofolate and flavin adenine dinucleotide cofactors, binds to the distorted region of DNA. The enzyme uses light energy—specifically, the absorption of a photon by the N, N lO-methenyltetrahydrofolate coenzyme—to form an excited state that cleaves the dimer into its original bases. 

Cryptochrome genes have been found in many organisms. In the fly Drosophila cryptochrome appears to interact directly with the clock proteins that control the circadian cycle. Most important are products of two genes per (period) and tim (timeless). They are helix-loop-helix DNA binding proteins that form heterodimers, are translocated to the nucleus, and repress their own transcription. Morning light leads to a rapid disappearance of e TIM protein. The cryptochrome CRY appears to react directly with TIM to inactivate it. However, details remain to be learned. " The circadian clock mechanism appears to be universal and the cryptochrome-2 mcryl gene) appears to function in the mouse. A human cDNA clone was found to have a 48% identity with a relative of cryptochromes, the (6-4) photolyase of Drosophila. 

As expected, the Z-principal axis of g is oriented perpendicular to the 71-plane of the flavin ring. Analyses of EPR and ENDOR data revealed angles of (—29 4)° and (—14 2)° between the X axis of g and the N5-H5 (or N5-D5) bond in (6 ) photolyase [38] and cyclobutane pyrimidine dimer (CPD) photolyase [28, 30], respectively. The factors that cause the reorientation of the X and Y axes of g of a neutral flavin radical in the two highly homologous cofactor binding pockets of CPD photolyase and (6 ) photolyase remain elusive. Also, the orientations of the principal axes of g relative to the molecular frame of the isoalloxazine ring of a flavin anion radical still need to be determined experimentally. 

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