Heterologous expression and Purification of Cry1.

<p><b>A</b>) Expression of Cry1 in <i>E.coli</i> 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>B</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.</p

Photoreactivation assay <i>Trichoderma reesei</i>.

<p><b>A</b>) 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>B</b>) Colonies of the experiment described in <b>A</b> 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.</p

Conservation of Promoter Melting Mechanisms in Divergent Regions of the Single-Subunit RNA Polymerases

The single-subunit RNA polymerases make up a widespread family of proteins found in phage, mitochondria, and chloroplasts. Unlike the phage RNAPs, the eukaryotic RNAPs require accessory factors to melt their promoters and diverge from the phage RNAPs in the regions where functions associated with promoter melting in the latter have been mapped, suggesting that promoter melting mechanisms in the eukaryotic RNAPs diverge from those in the phage enzymes. However, here we show that an element in the yeast mitochondrial RNAP, identified by sequence alignment with the T7 phage RNAP, fulfills a role in promoter melting similar to that filled by the T7RNAP “intercalating hairpin”. The yeast mitochondrial RNAP intercalating hairpin appears to be as important in promoter melting as the mitochondrial transcription factor, MTF1, and both a structurally integral hairpin and MTF1 are required to achieve high levels of transcription on a duplex promoter. Deletions from the hairpin also relieve MTF1 inhibition of promoter escape on premelted promoters, likely because such deletions disrupt interactions with the upstream edge of the transcription bubble. These results are consistent with recent structural and functional studies of human mitochondrial RNAP and further reveal the surprising extent of mechanistic conservation between the eukaryotic and phage-encoded members of the single-subunit RNAP family

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

<p>Analysis of <i>cry1</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100625#pone.0100625-Tamura1" target="_blank">[34]</a>.</p

Molecular analysis of <i>cry1</i> mutant and overexpressing strains.

<p>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. <b>A</b>) Schematic representation of Δ<i>cry1</i> genomic locus, parental genomic locus <i>cry1</i> and pOE<i>cry1</i>, digested with <i>Sma</i>I enzyme. <b>B</b>) Southern blot analysis of the parental strain (QM9414), overexpressing (OE<i>cry1</i>) and <i>cry1</i> mutant (Δ<i>cry1</i>). <b>C</b>) Schematic representation of replacement <i>cry1::hph</i>, digested with <i>Nco</i>I enzyme. <b>D</b>) Southern blot analysis of the parental strain (QM9414) and <i>cry1</i> mutant (Δ<i>cry1</i>). <b>E</b>) Schematic representation of parental genomic locus of <i>cry1</i> and the pOE<i>cry1</i>, digested with <i>EcoR</i>I enzyme. <b>F</b>) Southern blot analysis of the parental strain (QM9414) and overexpressing (OE<i>cry1</i>) strains. <b>G</b>) Schematic representation of pOE<i>cry1</i> and the parental genomic locus of <i>cry1</i>, digested with <i>Sal</i>I enzyme. <b>H</b>) Southern blot analysis of the parental strain (QM9414), overexpressing (OE<i>cry1</i>) and <i>cry1</i> mutant (Δ<i>cry1</i>) strains.</p

ΔTPR2 bypasses 8oxoG, but not an abasic site.

<p>Lesion bypass of EhDNApolB2 (lanes 1 to 14) and ΔTPR2 (lanes 15 to 28). The time course primer extension is described as in material and methods using equal amounts of DNA polymerases and 100 µM dNTPs. After incubation times of 2.5, 5, 10 and 20 minutes the primer extension reactions were stopped and run onto a 15% denaturing polyacrylamide gel.</p

Binding of Cry1 to DNA.

<p>EMSA Assay using: non-damage oligomers <b>A</b>) and 6–4 PP oligomers <b>B</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.</p

TPR2 is responsible of lesion bypass extension opposite an abasic site.

<p>Lesion bypass of wtEhDNApolB2 (lanes 1 to 5 and 11 to 15) and ΔTPR2 (lanes 6 to 10 and 16 to 20) extending from a primer containing a 3′OH purine or a pyrimidine opposite an abasic site. A primer containing a 3′OH dAMP (lanes 1 to 10) or dCMP (11 to 20) opposite an abasic site was subject to a time course primer extension reaction from 2.5 to 20 minutes using equal amounts of DNA polymerases and 100 µM dNTPs. The reaction products were run onto a 15% denaturing polyacrylamide gel.</p

Fidelity of translesion DNA synthesis of EhDNApolB2.

<p>Translesion bypass fidelity of EhDNApolB 20 nM of exonuclease deficient EhDNApolB were incubated with 1 nM of a set of substrates containing several DNA lesions. The reactions were carried out with four dNTPs or single dNTP addition. The dNTPs were present at a concentration of 15 µM. Samples were taken at 2.5 minutes, stopped with 50 mM EDTA and 90% formamide and run onto a 18% denaturing polyacrylamide gel electrophoresis for their analysis by phosphorimagery. (A) Control thymine (lanes 1 to 5), 8-oxo guanosine (lanes 6 to 10), and abasic site (lanes 11 to 15). (B) 5 S-6R and 5R-6S thymine glycol (lanes 1 to 5 and 6 to 10 respectively). The upper arrow depicts the length of the final product substrate and the bottom arrow indicates the used primer.</p

Comparative sequence analyses.

<p>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 <i>T. reesei</i> (Cry1 Tr), 6–4 photolyase <i>A. thaliana</i> (6–4 At), 6–4 photolyase <i>D. melanogaster</i> (6–4 Dm), 6–4 photolyase <i>X. leavis</i> (6–4 Xl) and Phl1 <i>C. zeae-maydis</i> (6–4 Czm). <b>B</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.</p