The Trichoderma reesei Cry1 protein is a member of the cryptochrome/photolyase family with 6-4 photoproduct repair activity

Abstract

DNA-photolyases use UV-visible light to repair DNA damage caused by UV radiation. The two major types of DNA damage are cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP), which are repaired under illumination by CPD and 6-4 photolyases, respectively. Cryptochromes are proteins related to DNA photolyases with strongly reduced or lost DNA repair activity, and have been shown to function as blue-light photoreceptors and to play important roles in circadian rhythms in plants and animals. Both photolyases and cryptochromes belong to the cryptochrome/photolyase family, and are widely distributed in all organisms. Here we describe the characterization of cry1, a member of the cryptochrome/photolyase protein family of the filamentous fungus Trichoderma reesei. We determined that cry1 transcript accumulates when the fungus is exposed to light, and that such accumulation depends on the photoreceptor Blr1 and is modulated by Envoy. Conidia of cry1 mutants show decreased photorepair capacity of DNA damage caused by UV light. In contrast, strains over-expressing Cry1 show increased repair, as compared to the parental strain even in the dark. These observations suggest that Cry1 may be stimulating other systems involved in DNA repair, such as the nucleotide excision repair system. We show that Cry1, heterologously expressed and purified from E. coli, is capable of binding to undamaged and 6-4PP damaged DNA. Photorepair assays in vitro clearly show that Cry1 repairs 6-4PP, but not CPD and Dewar DNA lesions.

Figure 1. Cry1 is closely related to the cryptochrome/6-4 photolyase family.

Analysis of cry1 based on multiple sequence alignments with some members of the cryptochrome/photolyase family. In blue cryptochrome/6-4 photolyase family, in green DASH cryptochromes, in red CPD photolyases and black bacterial cryptochromes and photolyases, is shown and the NCBI sequence identifier (gi) for each protein. The evolutionary history was inferred using the Minimum Evolution method. The optimal tree with the sum of branch length  = 15.50943863 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. The Neighbor-joining algorithm was used to generate the initial tree. The analysis involved 54 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 300 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 .

Figure 2. Comparative sequence analyses.

A) Sequence alignment among 6–4 photolyases and Cry1. Conserved (white on blue) and similar (black on gray) aminoacids are high-lighted; red circles indicate the tryptophan triad; amino acids binding FAD are indicated by orange squares (conserved) and green squares (similar), as well as the histidines needed for photorepair (black stars). Black circles indicate the non-conserved amino acids in the FAD binding region. Cry1 T. reesei (Cry1 Tr), 6–4 photolyase A. thaliana (6–4 At), 6–4 photolyase D. melanogaster (6–4 Dm), 6–4 photolyase X. leavis (6–4 Xl) and Phl1 C. zeae-maydis (6–4 Czm). B) Homology model of Cry1. The DNA photolyase and FAD domains are represented as ribbon and colored in cyan and blue respectively. Catalytic histidines 406 and 410 are in a ball-stick representation, the tryptophan triad and FAD are represented as spheres. The non-conserved C-terminal extension of Cry1 was left from the model.

Figure 3. Expression of cry1 in response to blue light.

Analysis of the expression of cry1 induced by a pulse of blue light (1200 µmol m⁻²) or upon exposure to constant light (fluence: 3.6 µmol m⁻² s⁻¹) for 72 h growth, as indicated. Transcript levels of the cry1 gene were determined by Northern blot analysis of the parental strain QM9414, Δenv1, Δblr1 strains. The gpd gene was used as loading control in the different conditions.

Figure 4. Molecular analysis of cry1 mutant and overexpressing strains.

Ten micrograms of genomic DNA were digested, separated on 1% agarose gel and hybridized with the probe indicated in the scheme. The position for each restriction enzyme and the sizes of the DNA fragments generated as indicated in each diagram. A) Schematic representation of Δcry1 genomic locus, parental genomic locus cry1 and pOEcry1, digested with SmaI enzyme. B) Southern blot analysis of the parental strain (QM9414), overexpressing (OEcry1) and cry1 mutant (Δcry1). C) Schematic representation of replacement cry1::hph, digested with NcoI enzyme. D) Southern blot analysis of the parental strain (QM9414) and cry1 mutant (Δcry1). E) Schematic representation of parental genomic locus of cry1 and the pOEcry1, digested with EcoRI enzyme. F) Southern blot analysis of the parental strain (QM9414) and overexpressing (OEcry1) strains. G) Schematic representation of pOEcry1 and the parental genomic locus of cry1, digested with SalI enzyme. H) Southern blot analysis of the parental strain (QM9414), overexpressing (OEcry1) and cry1 mutant (Δcry1) strains.

Figure 5. Analysis of the transformants for growth and expression in response to blue light.

A) Growth of transformants on PDA under constant light and dark for 72 h, fluence: 3.6 µmol m⁻² s⁻¹. B) Analysis of the expression of cry1 in transformants after exposure to a 5 min pulse of blue light (1200 µmol m⁻²). Transcript levels of the cry1 gene were determined by Northern blot analysis and the gpd gene was used as loading control.

Figure 6. Photoreactivation assay Trichoderma reesei.

A) Two hundred conidia of the strain indicated at the left of the figure were placed on PDA, and irradiated or not with UV light at 350 J m⁻², then incubated at 28°C for 18 h in a chamber with white light or kept in the darkness, as indicated. The images were taken at a 20X amplification with a binocular microscope. B) Colonies of the experiment described in A were counted and the results plotted as percent survival for each condition in relation to the control non-irradiated with UV light. Bars indicate standard deviation from two independent experiments. The statistical analysis included one-way ANOVA with a significance level of p<0.05. An asterisk indicates that strains are significantly different from the QM9414 strain in each treatment.

Figure 7. Heterologous expression and Purification of Cry1.

A) Expression of Cry1 in E.coli SY2 (a DNA repair-defective strain) and purification using a HIS-Trap FF column. 1. Non induced; 2. Induced with 0.1 mM of IPTG; 3. Insoluble fraction; 4. Cell free extract; 5. Flow through a nickel column; 6. Fraction of unbound proteins; 7. Fraction eluted with Imidazole 500 mM. B) Purification of Cry1 using a monoQ anion exchange column eluting with a NaCl gradient. 1. Flow through a monoQ column. 2–8. Fractions eluted with NaCl 50, 100, 200, 300, 400, 500, and 600 mM, respectively. MW. Molecular weight marker.

Figure 8. Binding of Cry1 to DNA.

EMSA Assay using: non-damage oligomers A) and 6–4 PP oligomers B). An arrow preceded by a C indicates the migration of oligomer-Cry1 complexes. 1 nM oligo-labelled, Cry1: 0, 4.23, 8.45, 12.68 and 25.35 µM respectively in lines 1–5.

Figure 9. In vitro Photorepair Assay.

Cry1 (7 µM) was incubated with 10 nM labeled oligonucleotides for 20 min under constant white light for photoactivation. After photoreactivation, the DNA was digested with MseI and separated on a 10% polyacrylamide gel. The digested product with MseI indicates photorepair of the substrate: A) Undamaged oligomer. B) CPD oligomer. C) Dewar oligomer. D) 6–4 PP oligomer. (+) Indicates presence and (−) absence of the enzyme indicated at the left.