DNA PHOTOLYASE FROM THE FUNGUS <i>NEUROSPORA CRASSA</i>. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH • ) induces an electron transfer over Ϸ15 Å from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH • 4 W382 4 W359 4 W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH • decayed with a time constant Ϸ 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived ( Ϸ 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH • is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield (Ϸ0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin͞neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH • is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH • ( Ϸ 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH • in WT and from W359 to excited FADH • in W382F mutant photolyase were estimated and related to the donor-acceptor distances.

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“…The final preparation was estimated to be Ͼ98% pure. E. coli WT photolyase was overexpressed and purified as described (26).…”

Section: Methodsmentioning

confidence: 99%

Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation

Byrdin

Eker

Vos

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

100

150

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“…The photolyase from the cyanobacterium A. nidulans [39] was investigated as a member of microbial deazaflavin-and flavin-containing photolyases (class I/type II). The photolyase from the fungus N. crassa [40] was used as a representative member of the class I/type I, flavin/methenyltetrahydrofolate-containing photolyases. Both enzymes feature a strong sequence homology with the well studied E. coli enzyme.…”

Section: Introductionmentioning

confidence: 99%

Synthesis, Crystal Structure, and Enzymatic Evaluation of a DNA-Photolesion Isostere

1998

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Nucleotide analogues are useful tools for the investigation of interactions between DNA-binding proteins and DNA at a molecular level. Herein we describe the synthesis of the DNA-lesion analogue 2, which is required to determine the extent to which specific phosphodiesters in the DNA backbone contribute to the recognition of cyclobutane pyrimidine dimer DNA lesion by the dimer-specific repair enzymes DNA photolyases or T4-endonuclease V. The analogue 2 is a close structural mimic of cyclobutane pyrimidine dimers like 1, which are the major lesions induced upon irradiation of cells with UV light. Instead of the negatively charged phosphate link in 1, analogue 2 contains an uncharged but isosteric formacetal moiety. The analysis of this and other phosphodiester contacts is hoped to provide insight into the lesion recognition process, which is currently believed to require the flipping of the lesioned base out of the DNA double helix. The lesion analogue 2 is synthetically available in large quantities, which allowed us to establish a new, fast and sensitive DNA photolyase assay. A precise X-ray crystal structure analysis of the DNA-lesion analogue 2 is also presented. The structure underlines the isosteric character of 2 and reveals, in combination with the only other available X-ray crystal structure determined from a thymine-dimer triester analogue, interesting structural features of cyclobutane pyrimidine dimer lesions. We describe the incorporation of the lesion analogue 2 into oligonucleotides by using standard phosphoramidite chemistry. Initial enzymatic repair studies are reported with three different types of DNA photolyases. These studies show that the lesion analogue 2 is rapidly repaired by photolyases from Anacystis nidulans, Neurospora crassa and from the marsupial Potorous tridactylis. The enzymatic investigations indicate that all photolyases, including enzymes from higher organisms (P. tridactylis) accept the formacetal dimer as a lesion substrate and therefore could possess a similar DNA-lesion recognition process, in which the interaction with the central phosphate unit is only of limited importance.

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“…Although A. nidulans also has an oscillator system (Greene et al, 2003), it does not show obvious circadian rhythmicity. More than a decade ago, the N. crassa photolyase was shown to have photorepair activity in Escherichia coli, but its role in other processes of the fungal development remained elusive (Eker et al, 1994). It was recently shown that the Trichoderma atroviride photolyase PH1 autoregulates its expression, which suggests that fungal photolyases might possess additional regulatory functions (Berrocal-Tito et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

More Than a Repair Enzyme:Aspergillus nidulansPhotolyase-like CryA Is a Regulator of Sexual Development

Bayram

Biesemann

Krappmann

et al. 2008

MBoC

127

116

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Cryptochromes are blue-light receptors that have presumably evolved from the DNA photolyase protein family, and the genomes of many organisms contain genes for both types of molecules. Both protein structures resemble each other, which suggests that light control and light protection share a common ancient origin. In the genome of the filamentous fungus Aspergillus nidulans, however, only one cryptochrome/photolyase-encoding gene, termed cryA, was identified. Deletion of the cryA gene triggers sexual differentiation under inappropriate culture conditions and results in upregulation of transcripts encoding regulators of fruiting body formation. CryA is a protein whose N-and C-terminal synthetic green fluorescent protein fusions localize to the nucleus. CryA represses sexual development under UVA 350-370 nm light both on plates and in submerged culture. Strikingly, CryA exhibits photorepair activity as demonstrated by heterologous complementation of a DNA repair-deficient Escherichia coli strain as well as overexpression in an A. nidulans uvsB⌬ genetic background. This is in contrast to the single deletion cryA⌬ strain, which does not show increased sensitivity toward UV-induced damage. In A. nidulans, cryA encodes a novel type of cryptochrome/photolyase that exhibits a regulatory function during light-dependent development and DNA repair activity. This represents a paradigm for the evolutionary transition between photolyases and cryptochromes. INTRODUCTIONCryptochromes (CRYs) are blue-light receptors that regulate growth, development, and the circadian clock in higher eukaryotes and that are believed to have evolved from the DNA photolyase protein family (Daiyasu et al., 2004;Lin and Todo, 2005). Photolyases, in contrast, repair UV-induced DNA damage by using a mechanism referred to as photorepair. They absorb light in the blue spectrum and transfer an excited electron from the cofactor FAD to an enzyme-bound cyclobutane pyrimidine dimer (CPD), which is thereby cleaved (Sancar, 1990(Sancar, , 1994Sancar, 1994). Cryptochromes are characterized by an N-terminal photolyase-related (PHR) region without significant photorepair activity and by a C-terminal domain of varying length, which is absent in members of the CRY-DASH subfamily (Daiyasu et al., 2004). The exact function of the CRY-DASH proteins was elusive; however, recent data imply that they are actually photolyases that specifically repair CPDs in single-stranded DNA (Selby and Sancar, 2006). The PHR domain is the most conserved region of the CRY proteins and contains the cofactor FAD required for electron transfer reactions. In Xenopus and Drosophila, the PHR domain is physiologically active even in the absence of the C-terminal domain. The C-terminal domain is important for either localization or protein stability, and its expression in Arabidopsis results in constitutive growth (Lin and Todo, 2005). Plant CRYs are phosphorylated under illumination, and gene expression control ranges from protein interactions to direct chromatin interactions (Shalitin et al....

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DNA PHOTOLYASE FROM THE FUNGUS NEUROSPORA CRASSA. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES

Cited by 44 publications

References 44 publications

Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation

Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation

Synthesis, Crystal Structure, and Enzymatic Evaluation of a DNA-Photolesion Isostere

More Than a Repair Enzyme:Aspergillus nidulansPhotolyase-like CryA Is a Regulator of Sexual Development

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