PMF determination for carbinolamine formation with water-mediated proton transfer.

<p>The work vs. CV functions for each of 30 SMD replicates are given in color in Panel A as is their Jarzynski average (solid black line). For clarity, only the Jarzynski average, the bias corrected Jarzynski average (solid red line) the square root of the mean square error (MSE as defined in Gore, <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031377#pone.0031377-Gore1" target="_blank">[24]</a>, dashed red lines), and the definition of the CV are shown in Panel B. Note that CV steering is from 8.92 Å to 3.52 Å, i.e., right to left for the formation of the carbinolamine. The large scatter in “barrier height” seen for the individual work functions and the scatter in the work values at the end of the simulation (left axis) are normal and expected for these kinds of methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031377#pone.0031377-Xiong1" target="_blank">[39]</a>. Such barrier heights apply only to the pathway followed in that trajectory, and the individual work functions are not good estimates of the pathway G function for the reaction. The purpose of the exponential averaging in the Jarzynski algorithm is to properly weight the individual work functions so that functions with large peaks contribute only to a degree appropriate to their probability of occurrence. The estimate of G along the reaction pathway should use the Jarzynski average or some other comparable function such as its bias corrected version (the better choice) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031377#pone.0031377-Gore1" target="_blank">[24]</a>.</p

Carbinolamine dehydration with participation of an intermediate water.

<p>Panel A shows proton transfers associated with the carbinolamine dehydration in this unforced reaction mediated via a water intermediate. Panel B shows the transfer of the water proton to the carbinolamine oxygen to form the product water. Panel C shows trajectory plots for the oxygen departure (dehydration) and the product separation (bonding) distance (product water formation). The progress of the simulation is shown color coded as indicated in the palette. The identities of the atoms in quantum region functional groups are shown in color: black, red or blue. The time between samples of the trajectory was 2.5 ps.</p

Stereogram of a typical initial quantum region for carbinolamine formation mediated by an active site water.

<p>The large purple “atoms” are the link hydrogens, proxies for the classical mechanics sites covalently bonded to the quantum region. The atomic coordinates for this figure were taken directly from one of the Protein Data Bank (PDB) files for the quantum atoms written at the beginning of each QM/MM run. Molecular graphics in this and other similar figures were prepared with MOLSCRIPT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031377#pone.0031377-Kraulis1" target="_blank">[37]</a> and Raster3D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031377#pone.0031377-Merritt1" target="_blank">[38]</a>.</p

Stereogram of the quantum region for direct proton transfer carbinolamine formation without water involvement.

<p>The atom names and distances referenced in Panel B of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031377#pone-0031377-g005" target="_blank">Fig. 5</a> are defined. The atoms are colored and the QM/MM region atomic coordinates are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031377#pone-0031377-g002" target="_blank">Fig. 2</a>.</p

PMF determination for carbinolamine formation by direct proton transfer without involvement of an intermediate water.

<p>The work vs. CV functions for each of 30 SMD replicates are given in color in Panel A as is their Jarzynski average (solid black line). For clarity only the Jarzynski average, the bias corrected Jarzynski average the square root of MSE, and CV definition are shown in Panel B, and is similar to panel B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031377#pone-0031377-g003" target="_blank">Fig. 3</a>. As in that figure, the steered direction for carbinolamine formation is right to left.</p

Reactions catalyzed by lyase-capable BER glycosylases.

<p>This diagram illustrates the possible reaction pathways catalyzed by these enzymes. The proportions of abasic sites and – and –elimination products in the product spectra depend on the intrinsic chemistry of the imine intermediates and the relative rates of their (1) hydrolytic turnover, e.g., <b>Schiff base intermediate</b><b>E–Abasic site</b> and (2) conversion to “downstream” products, e.g., <b>Schiff base intermediate</b><b>E–SSB (covalent)</b>. This report mainly investigates the reactions corresponding to <b>E–Abasic site intermediate</b><b>Schiff base</b> in the figure: the collapse of the amine nitrogen onto the carbonyl carbon to form the carbinolamine intermediate followed by its dehydration. These reactions are outlined by the rectangular box in the figure. <b>E–DNA</b> may represent the enzyme bound at the location of either an abasic site, a pyrimidine photodimer or a lyase product of the – or –elimination type.</p

Structure and purity of stereoisomeric deoxyadenine <i>N</i>⁶-BPDE adducts.

<p>(A) Structure of the (+) and (-) <i>anti-trans</i> BPDE that are covalently linked to adenine <i>N</i>⁶ placed at the third position of N-ras codon 61 within an 11-mer oligodeoxynucleotide. (B) Autoradiogram of oligodeoxynucleotides that were ³²P-labeled at the 5´-terminus and subjected to electrophoresis through 15% polyacrylamide gels. Lanes U, S and R represent unadducted, (<i>+</i>)<i>-anti-trans</i>- and <i>(-)-anti-trans</i>-BPDE-adducted templates, respectively.</p

Primer extension analysis on unadducted and BPDE-adducted primers with alternate DNA polymerases.

<p> Polymerase extensions of 27t/11p^* or 11p were performed as described in MATERIALS and METHODS. In all cases, 10 fmol of enzyme was used. The primer to polymerase ratio was 5. U, S, and R represent unadducted, (+) and (-)<i>-anti-trans</i>-BPDE-adducted primers, respectively. The unadducted and adducted primers have different electrophoretic mobilities that result in products of different mobilities. Panels represent primer extension assays in the presence of the respective polymerase: HIV-1 RT(RT), DNA polymerase β (pol β), Sequenase (Seq), KF exo^- (KF exo^-). The template sequence on the right coincides with full-length products that can be obtained by utilizing an 11-mer primer. The formation of truncated products is unique only to the <i>R</i>-adducted primer extended by HIV-1 RT. In some instances, non-templated blunt-end additions are observed (e.g., HIV-1 RT).</p

Evidence for the Involvement of DNA Repair Enzyme NEIL1 in Nucleotide Excision Repair of (5′<i>R</i>)- and (5′<i>S</i>)-8,5′-Cyclo-2′-deoxyadenosines

The DNA repair enzyme NEIL1 is a DNA glycosylase that is involved in the first step of base excision repair (BER) of oxidatively induced DNA damage. NEIL1 exhibits a strong preference for excision of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA with no specificity for 8-hydroxyguanine (8-OH-Gua). In this study, we report on the significant accumulation of (5′<i>R</i>)-8,5′-cyclo-2′-deoxyadenosine (<i>R</i>-cdA) and (5′<i>S</i>)-8,5′-cyclo-2′-deoxyadenosine (<i>S</i>-cdA) in liver DNA of <i>neil1</i>^{<i>−/−</i>} mice that were not exposed to exogenous oxidative stress, while no accumulation of these lesions was observed in liver DNA from control or <i>ogg1</i>^{<i>−/−</i>} mice. Significant accumulation of FapyGua was detected in liver DNA of both <i>neil1</i>^{<i>−/−</i>} and <i>ogg1</i>^{<i>−/−</i>} mice, while 8-OH-Gua accumulated in <i>ogg1</i>^{<i>−/−</i>} only. Since <i>R</i>-cdA and <i>S</i>-cdA contain an 8,5′-covalent bond between the base and sugar moieties, they cannot be repaired by BER. There is evidence that these lesions are repaired by nucleotide excision repair (NER). Since the accumulation of <i>R</i>-cdA and <i>S</i>-cdA in <i>neil1</i>^{<i>−/−</i>} mice strongly points to the failure of their repair, these data suggest that NEIL1 is involved in NER of <i>R</i>-cdA and <i>S</i>-cdA. Further studies aimed at elucidating the mechanism of action of NEIL1 in NER are warranted

Relative dissociation rate constants from adducted or unadducted DNA.

<p>Kinetic analyses of the extension of labelled primer (33t/29t) when 2.5 fmol of HIV-1 RT was preincubated with unadducted (open circles), 27t/11p C₁₀<i>R</i> (closed circles)- or C₁₀<i>S</i> (closed squares)-adducted 11-mer primers (27t/11p^*). U, S, and R represent unadducted, (+) and (-) <i>anti-trans-</i>BPDE adducted templates, respectively.</p