Phylogenetic relationships among the PKA catalytic subunit homologs in chordates.

<p>The Cα and Cβ paralogs are a result of a gene duplication in a common ancestor of vertebrates. Subsequent duplications of Cα and Cβ in a teleost fish ancestor have resulted in four PKA catalytic subunits in these organisms. The Bayesian inference tree is based on the nucleotide sequences (codon positions 1 and 2 only, GTR+Γ+I model) of exons 2 to 10 which corresponds to a multiple sequence alignment with no gaps. The phylogram is shown with estimated branch lengths proportional to the number of substitutions at each site, as indicated by the scale bar. The arthropod fruit fly (<i>D. melanogaster</i>) and the echinoderm sea urchin (<i>S. purpuratus</i>) have been set as outgroups. Bayesian posterior probabilities are shown for each node. The topology of a maximum likelihood (ML) tree generated with the same data set and model was identical to the Bayesian inference tree. ML bootstrap values are shown for selected nodes (1000 replications). The sequences of human and mouse PKA Cα and Cβ and the homologs from amphioxus (<i>B. floridae</i>), zebra finch (<i>T. guttata</i>), chicken (<i>G. gallus</i>), the frog <i>X. tropicalis</i>, the lizard <i>A. carolinensis</i>, medaka (<i>O. latipes</i>), the pufferfish <i>T. rubripes</i>, and stickleback (<i>G. aculeatus</i>) are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060935#pone.0060935.s003" target="_blank">Materials and Methods S1</a>. The <i>X. tropicalis</i> Cα and <i>A. carolinensis</i> Cβ are incorrectly placed (See discussion and Fig. 3).</p

The identity of eleven amino acids in the protein chain may define the Cα and Cβ branches of PKA catalytic subunits.

<p>Our full set of PKA catalytic subunits (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060935#pone.0060935.s003" target="_blank">Materials and Methods S1</a>) from bony fishes and tetrapods, comprising 27 Cα and 33 Cβ, was employed to identify eleven amino acid positions that together may be used to classify a PKA catalytic subunit as belonging to one of the two branches. The sequence logos define the PKA Cα and Cβ clades within the <i>Teleostomi</i>, which includes the familiar classes of bony fishes, birds, mammals, reptiles, and amphibians. We find invariable Gln35, Thr37, Glu64, Gly66, His68, Ser109 and Glu334 in Cα and invariable Asp42, Gln67, and Arg319 in Cβ (Cα1/Cβ1 numbering). The residues in the corresponding positions in human Cα1 and Cβ1 are also shown.</p

The Bayesian inference trees for vertebrate PKA Cα and Cβ both closely reflects the evolutionary relationships among these organisms.

<p><b>A</b> Phylogenetic analysis of Cα orthologs resulted in a tree that was rooted with human and mouse Cβ as outgroups. The tree was based on the nucleotide sequences of exons 2 to 8 (all codon positions, GTR+Γ+I model). <b>B</b> Phylogenetic analysis of Cβ orthologs was performed employing nucleotide sequence data (all codon positions, exons 2 to 10, GTR+Γ+I model). The resulting tree was rooted with human and mouse Cα as outgroups. In both trees, branch lengths are shown as substitutions per site, with scale indicated by the scale bars. Bayesian posterior probabilities are given for each node and ML bootstrap values (1000 replications) are shown for selected nodes where the clades are identical in the Bayesian and ML analysis. In addition to organisms found in Fig. 2, representative sequences from the following species were included: eutherian mammals rhesus macaque (<i>M. mulatta</i>), tarsier (<i>T. syrichta</i>), dog (<i>C. familiaris</i>), horse (<i>E. caballus</i>), pig (<i>S. scrofa</i>), cow (<i>B. taurus</i>), rat (<i>R. norvegicus</i>), and hamster (<i>C. griseus</i>), marsupial mammals wallaby (<i>M. eugenii</i>) and opossum (<i>M. domestica</i>), the frog <i>X. laevis</i>, the pufferfish <i>T. nigroviridis</i> and Atlantic salmon (<i>S. salar</i>). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060935#pone.0060935.s003" target="_blank">Materials and Methods S1</a> for the sequence data.</p

Structural model with proposed active site and lesion recognition pocket of AlkD

<p><b>Copyright information:</b></p><p>Taken from "Structural insight into repair of alkylated DNA by a new superfamily of DNA glycosylases comprising HEAT-like repeats"</p><p></p><p>Nucleic Acids Research 2007;35(7):2451-2459.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1874660.</p><p>© 2007 The Author(s)</p> The model contains residues 11 through to 226 and is lacking α-helix α1 and the 11 C-terminal residues (predicted to be disordered). () Cartoon rendering of the protein which comprises 13 α-helices contributing to the six repeats in . () APBS () calculated electrostatic potential mapped onto the protein surface (red = negative, white = neutral and blue = positive) showing the 20–25 Å wide, positively charged, putative DNA binding groove. () Amino acid residue conservation in 43 AlkD homologs mapped onto the space filling representation of the model generated with ConSurf (). The scale extends from magenta (highly conserved), through white to cyan (highly variable). There is a nest of conserved residues in the putative DNA binding groove, and several conserved basic amino acid residues (Arg and Lys) are sited along the upper and lower edge of the groove. () Stereo view of a close-up of the highly conserved nest shows the eight conserved residues that were mutated by site-directed mutagenesis. The catalytic activity and MMS sensitivity of the resulting mutants were determined (). The eight conserved residues have identical geometry in the experimental structure 2B6C. The orientation of the protein in space is identical in all panels. The model is also available as Supplementary Video 1 online

Mtb XPB unwinding activity on bubble, D- and R-loop structures.

<p>The oligos used in this experiment are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036960#pone.0036960.s008" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036960#pone.0036960.s009" target="_blank">S2</a>. i) bubble substrate; ii) D-loop with fully complementary invading strand with no tail; iii) D-loop with 3′ tail; iv) R-loop with 3′ tail; v) D-loop with 5′ tail; vi) R-loop with 5′ tail. Lane 1. no enzyme; lane 2. heat-denatured substrate; lanes 3–5. 500, 1000 and 2000 nM Mtb XPB, respectively. Open arrow- complex DNA substrates; closed arrow- unwound products.</p

DNA strand annealing activity of Mtb XPB.

<p>A) Enzyme concentration-dependent strand annealing activity of Mtb XPB. Labeled C80 oligo (1 nM) incubated with unlabeled G80 oligo (1 nM) in the absence of ATP and increasing concentration of Mtb XPB for 15 min. Lane 1. no enzyme; lanes 2–10. Mtb XPB [nM] 25, 50, 100, 200, 400, 800, 1600, 2000 and 5000 respectively; lanes 11–13. mock dilution: 1∶1, 1∶10, 1∶100, respectively; lane 14. M- duplex marker (80 bp). (B) Unlabeled G80 oligo concentration-dependent strand annealing activity of Mtb XPB. Labeled C80 oligo incubated for 15 min with increasing concentration of unlabeled G80 oligo and with/without 250 nM Mtb XPB in the absence of ATP. Lanes 1–6. reactions in the absence of Mtb XPB (-); lanes 7–11. reactions in the presence of Mtb XPB; lane 11. M- duplex marker (80 bp). C) Time course of strand annealing activity carried out at different time intervals in the absence of enzyme or in the presence of Mtb XPB [250 nM] or <i>E. coli</i> RecQ [10 nM]. M- duplex marker (80 bp). D) Quantitation of % reaction product at the indicated time points.</p

Titration of Mtb XPB unwinding activity.

<p>DNA unwinding activity was titrated in the presence of 1 nM forked DNA (T1+B1- a 30mer complementary region with non-complementary 30mer tails) and increasing concentrations of Mtb XPB. A) Representative gel analysis showing unwinding reaction products. Lanes: 1. no enzyme; 2. HD-heat-denatured substrate; 3. 250 nM; 4. 500 nM; 5. 1000 nM; 6. 2000 nM; 7. 3000 nM; 8. 4000 nM; 9. 5000 nM; 10. <i>E. coli</i> RecQ (10 nM); 11–13. mock dilutions, 1∶1, 1∶10 and 1∶100, respectively. B) Quantitation of unwinding activity of Mtb XPB. The average of 3 independent experiments and standard deviations (error bars) are shown.</p

Mtb XPB binds DNA substrates.

<p>Representative gels of resolved Mtb XPB-DNA binding mixtures using the DNA substrates given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036960#pone.0036960.s008" target="_blank">Table S1</a>. Increasing Mtb XPB concentrations were incubated with the indicated DNA substrate (100 pM) for 15 min on ice in EMSA buffer. i) ssDNA (T1), ii) duplex DNA (T1+B2), iii) bubble DNA ( T1+B3), iv) forked DNA (T1+B1). Lane 1. no enzyme; lanes 2–6. Mtb XPB [nM] 500, 1000, 2000, 4000 and 5000, respectively. v) 2000 nM Mtb XPB was incubated with ssDNA substrates containing 30 (B5, 1 & 2); 60 (T1, 3 & 4) and 80 (C80, 5 & 6) bases. Lanes 1, 3 and 5–reactions without enzyme; lanes 2, 4 and 6–reactions with enzyme. vi) 2000 nM Mtb XPB was incubated with 30 bp duplex (B5+B6, l & 2), 60 bp duplex (T1+B2, 3 & 4), forked DNA (T1+B1, 5 & 6), bubble DNA (T1+B3, 7 & 8) and 80 bp duplex (C80+G80, 9 & 10). Lanes 1, 3, 5, 7 and 9- reactions without enzyme; lanes 2, 4, 6, 8 and 10–reactions with enzyme.</p

Requirement of ATP hydrolysis for Mtb XPB unwinding activity.

<p>Labeled forked DNA substrate was incubated with Mtb XPB in the presence of 2 mM ATP, dATP, ATPγS, or ADP for 30 min at 37°C. Lane 1. no enzyme; lane 2. heat-denatured substrate; Lanes 3, 5, 7 and 9. 1000 nM Mtb XPB; Lanes 4, 6, 8 and 10. 2000 nM Mtb XPB; lanes 11–14. ‘mock’ preparation (1∶10 dilution); lane 15. reaction containing Mtb XPB (2000 nM) and without any nucleotide cofactors.</p

XPB domain organization and functional motifs.

<p>A) Domain organization showing the N- and C-terminal RecA-like helicase domains and the N-terminal domain unique to the XPB family of proteins. This latter domain is found in eukaryote, bacterial and some archaeal XPBs, but appears to be replaced by a shorter DNA damage recognition domain (DRD, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036960#pone.0036960-Fan1" target="_blank">[26]</a>) in <i>A. fulgidus</i> and a subset of archaea. Numbers indicate the approximate domain boundaries. B) Structural model of the Mtb XPB N-terminal RecA-like helicase domain (in light cyan) showing bound ADP (sticks) and a divalent cation (sphere), the Q-motif (colored red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036960#pone.0036960-Cordin1" target="_blank">[31]</a>, the RED motif (purple) conserved in the XPB family <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036960#pone.0036960-Fan1" target="_blank">[26]</a> and the helicase motifs I (orange), Ia (yellow), II (green) and III (blue). The hinge region linking the two RecA-like domains is shown in pink. C) Model of the Mtb XPB C-terminal RecA-like helicase domain (in yellow) with helicase motifs IV (red), V (green) and VI (blue). The hinge region is shown in pink. D) A multiple sequence alignment (MSA) of XPB homologs demonstrates that the N-terminal domain unique to the XPB family of proteins is present in eukaryotes, in yeast (<i>Saccharomyces cerevisiae</i> RAD25, NCBI Refseq identifier NP_012123) and human XPB/ERCC3 (NP_000113), in bacteria, <i>e.g. M. tuberculosis</i> H37Rv (NP_215376), <i>Propionibacterium acnes</i> (YP_003580697), <i>Bacillus tusciae</i> (YP_003589555) and <i>Treponema pallidum</i> (NP_218820) and in some archaea, including <i>Haloferax volcanii</i> (YP_003535766). Sequence numbering is given at the line ends. E) An MSA of the two RecA-like helicase domains of XPB from the three domains of life shows the location of classical helicase motifs, the hinge region linking the two domains, the Q- and RED-motifs and the flexible thumb motif (ThM, grey) that is unique to a subset of archaea including <i>A. fulgidus</i> (NP_069194). The coloring of the motifs is identical to panels B) and C).</p