EcoNth active fraction determination by gel-based and molecular accessibility assays

<p><b>Copyright information:</b></p><p>Taken from "Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates"</p><p></p><p>Nucleic Acids Research 2007;35(5):1601-1611.</p><p>Published online 8 Feb 2007</p><p>PMCID:PMC1865064.</p><p>© 2007 The Author(s).</p> 0, 50, 100, 200, 400 and 800 nM EcoNth was incubated for 30 min at 37°C with either 100 nM of 35DHU substrate ( and ) or 100 nM of 4L, 5L, 5R or N5R substrates ( and ) in the presence of either 50 mM sodium borohydride () or 50 mM sodium cyanoborohydride (). Completed 35DHU reactions were separated by 12% SDS-PAGE; results from phosphorimager analysis with calculated α values are plotted below each gel image. Completed 4L, 5L, 5R and N5R reactions were mixed with an equal volume of 200 nM DAPI solution, incubated for 5 min at room temperature, and fluorescence was detected at 340 nm excitation/460 nm emission. Relative fluorescence readings to the 0 nM EcoNth sample were used to determine the concentration of ES complex at each enzyme concentration. Error bars representing the standard deviation from three independent experiments are shown on all points where the error was larger than the body of the symbol. See the materials and methods section for further details of experimental design and analysis

NEIL1 and EcoNei active fraction determination by the molecular accessibility assay

<p><b>Copyright information:</b></p><p>Taken from "Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates"</p><p></p><p>Nucleic Acids Research 2007;35(5):1601-1611.</p><p>Published online 8 Feb 2007</p><p>PMCID:PMC1865064.</p><p>© 2007 The Author(s).</p> NEIL1, at concentrations of 50 and 100 nM, and EcoNei, at concentrations of 100 and 200 nM, were incubated with 100 nM 4L substrate in the presence of 50 mM sodium borohydride. Completed reactions were mixed with an equal volume of 200 nM DAPI solution, incubated for 5 min at room temperature, and fluorescence was detected at 340 nm excitation/460 nm emission. Relative fluorescence readings to the 0 nM enzyme sample were used to determine the concentration of ES complex using the equation ES = × (1 − RF) where RF is the relative fluorescence at each given enzyme concentration, as shown in the table (left). Plotted ES concentrations (right) were used to determine the α values by linear regression. Activity is simply defined as the percentage of determined α values (100 × α). NEIL1, solid circles; EcoNei, open circles

LSCs supply or stabilize residues that participate in enzyme-DNA interactions.

<p>Top) Amino acids side chains associated with LSC 1–6 are shown in the context of the protein backbone, DNA backbone, damaged nucleotide, opposite nucleotide, and Zn ion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Fromme1" target="_blank">[67]</a>. The green residues in both the structure (top) and the diagram (bottom) correspond to first-shell amino acids conserved in the entire family: R264 (contained in LSC6), N174 (stabilized by LSC1), and K60 (stabilized by LSC3/LSC2) stabilize the phosphate of the damaged base, and P2, E3 and are part of the catalytic residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Gilboa1" target="_blank">[97]</a>. The helix containing P2 and E3 may be stabilized by LSC2 as well. The enzyme everts the damage, and an intercalation loop (LSC4) fills the void and makes contact with the opposite base <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Coste1" target="_blank">[68]</a>. The damage itself in BaFpg1 is recognized by a recognition complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Fromme1" target="_blank">[67]</a>. Other important residues not included here include H74 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Gilboa1" target="_blank">[97]</a> and E6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Kropachev1" target="_blank">[48]</a>. A DNA binding residue not discussed in the literature corresponds to Tyr242 (part of LSC5).</p

The states of LSCs can be used to infer the Fpg/Nei family phylogeny.

<p>The most parsimonious protein phylogeny consistent with the states of the six LSCs is shown with the changes in LSCs annotated as red bars. The choice of the root results in one of its children (BaFpg1, BaFpg2) represents well the diversity of bacteria while the other represents plants, fungi, and metazoans.</p

Seven LSCs from the Fpg/Nei protein family.

<p>Seven LSCs from the Fpg/Nei protein family.</p

Multiple States of an LSC: Two solutions to the same problem.

<p>An LSC can have multiple states. A) State of LSC1 in the B. stearothermophilus MutM structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Fromme1" target="_blank">[67]</a>. N174 (in pink), part of the helix-two-turn-helix (H2TH) motif along with two other amino acids (including the key amino acid R264, in blue) functions in the orientation and kinking of the DNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Zharkov2" target="_blank">[70]</a>. K160 (blue) helps keep the proper arrangement between the zinc finger and the H2TH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Sugahara1" target="_blank">[69]</a>. B) Sequence logos for the each of the nine LSC1 amino acids in each of the three clades as well as MvNei1. Column headings indicate the aligned position in both the B. stearothermophilus MutM and E. coli Nei sequences. The sequence logos associated with 1R2Y K160 suggest that in three of the nine clades (BaFpg1, BaFpg2 and PFNei) the arrangement between the zinc finger and the H2TH is stabilized by a lysine in the same manner as in the B. stearothermophilus MutM protein. C) State of LSC1 in the E. coli Nei structure (62, PDB 1K3W). R171 hydrogen bonds to the other beta-sheet of the zinc-finger, presumably playing a role analogous to 1R2Y K160, which originates on a different helix. The sequence logos associated with R171 suggests that in six subfamilies (AcNei1 and AcNei2, PrNei and all vertebrate subfamilies), the arrangement between the zinc finger and the H2TH is maintained by an arginine or lysine in the same manner as in the E. coli Nei protein. For the subfamilies of BaFpg1 and PrNei, sites 160 and 266 are a type I, 174 and 264 are a type 0, and the rest are type II.</p

Example of structural clustering of type I sites.

<p>Type I sites, amino acids that shift in substitution rate among two clades (BaFpg1, and PFNei), are colored in green in the B. stearothermophilus MutM structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Fromme1" target="_blank">[67]</a> (1R2Y). Three structural clusters (LSCs) are shown, the (a) zinc finger (BaFpg1), zincless finger (PFNei); b) two highly conserved glycines on Fpg which mark the beginning and end of the recognition loop, and which have a higher rate on PFNei, suggesting that the loop does not perform the same role in recognition and c) a triad that stabilizes the DNA and the opposite base which allows for more variability on PFNei.</p

Rate variation does not differ dramatically between replicate Proteobacterium, Actinomycete, or Eukaryote organism tree topologies.

<p>Each column corresponds to one of the three organismal phylogenies. Each entry in a column (paired blue and green bars) represents an instance of the organismal phylogeny in the Fpg/Nei family protein phylogeny. The blue bars correspond to the number of substitutions from the last common ancestor (LCA) of each replicate tree to the present while the green bars correspond to the number of substitutions from the LCA of the phylogeny of replicate trees to the LCA of the each replicate tree.</p

Coefficient of Type I (above diagonal) and Type II (below diagonal) functional divergence for Fpg/Nei clades.

<p>Coefficient of Type I (above diagonal) and Type II (below diagonal) functional divergence for Fpg/Nei clades.</p

Substitution rates of individual aligned amino acid positions can differ between clades of orthologs.

<p>Substitution rates of individual aligned amino acid positions can differ between clades of orthologs from actinomycetes (left, Pearson correlation 0.47) or eukaryotes (right, 0.19). Each axis reflects amino acid variation rate in one of the replicate organism trees described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone-0025246-g004" target="_blank">Figure 4</a>. Each point is an aligned amino acid sequence position. Sites that have experienced a rate-shift (Type I) are green while those that exhibit an amino acid frequency-shift (Type II) are red.</p