formica_genotypes

formica_genotype

Effect of the linker length on the rate of the disulfide bond formation.

<p>(<b>A</b>) Pt-6 (C4) (open circles) and Pt-6 (C3) (filled circles); (<b>B</b>) AP-6 (C4) (open squares) and AP-6 (C3) (filled squares); (<b>C</b>) 64–6 (C4) (open triangles) and 64–6 (C3) (filled triangles). The dashed and continuous lines represent the decrease of the starting materials and the increase of the cross-linked products, respectively, and the standard errors are shown. The curve fitting was performed with the Origin 9.1 software, and the kinetic constants for the product formation are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117798#pone.0117798.t001" target="_blank">Table 1</a>.</p

HPLC analysis of disulfide bond formation in duplexes containing the cisplatin adduct.

<p>(<b>A</b>–<b>F</b>) Chromatograms of the duplexes, in which × represents cisplatin, after the reactions for the indicated length of time. The y-axis of each chromatogram was normalized. The cross-linked products and the sulfinic acid-containing oligonucleotides are indicated by an arrow and an asterisk, respectively, and the results of the DTT treatment of the products are shown in red. (<b>G</b>) Comparison of the product formation between GG-6 and Pt-6. The product peak areas in panels <b>B</b> and <b>E</b> were quantified.</p

Structures of the B-form (A) and cisplatin-induced bent (B) DNA duplexes.

<p>The duplexes with the PDB codes of 2K0V and 1A84 are shown. The distances between the C2’ atoms of the modified nucleosides were calculated using the PyMOL 1.7.1 software.</p

Disulfide bond formation dependent on the helix bending of DNA.

<p>(<b>A</b>) Schematic presentation of this study. Two mercaptoalkyl groups were attached to positions across the major groove of a duplex (left), and a disulfide bond was formed when helix bending occurred into the major groove (right). (<b>B</b>) The chemical structure of the thiol-tethered nucleoside. Another modified nucleoside bearing the 3-mercaptopropyl group was also used. (<b>C</b>–<b>E</b>) The chemical structures of the cisplatin adduct (<b>C</b>), the abasic site analog (<b>D</b>), and the (6–4) photoproduct (<b>E</b>).</p

HPLC analysis of disulfide bond formation in duplexes containing the (6–4) photoproduct.

<p>C4 and C3 represent the 4-mercaptobutyl and 3-mercaptopropyl groups, respectively. The y-axis of each chromatogram was normalized. The cross-linked products are indicated by an arrow, and the results of the DTT treatment of the products are shown in red.</p

Structures of the B-form (A) and cisplatin-induced bent (B) DNA duplexes.

<p>The duplexes with the PDB codes of 2K0V and 1A84 are shown. The distances between the C2’ atoms of the modified nucleosides were calculated using the PyMOL 1.7.1 software.</p

HPLC analysis of disulfide bond formation in duplexes containing the abasic site analog.

<p>(<b>A</b>, <b>B</b>, <b>C</b>, <b>E</b>, and <b>F</b>) Chromatograms of the duplexes, in which X represents the abasic site analog, after the reactions for the indicated length of time. C4 and C3 represent the 4-mercaptobutyl and 3-mercaptopropyl groups, respectively. The y-axis of each chromatogram was normalized. The cross-linked products and the sulfinic acid-containing oligonucleotides are indicated by an arrow and an asterisk, respectively, and the results of the DTT treatment of the products are shown in red. (<b>D</b>) Comparison of the product formation between AP-6 (C4) and AP·A-6 (C4). The product peak areas in panels <b>A</b> and <b>B</b> were quantified.</p

Effects of 5′,8-Cyclodeoxyadenosine Triphosphates on DNA Synthesis

Hydroxyl radicals generate a broad range of DNA lesions in living cells. Cyclopurine deoxynucleosides (CPUs) are a biologically significant class of oxidative DNA lesions due to their helical distortion and chemically stability. The CPUs on DNA are substrates for the nucleotide excision repair (NER) but not for base excision repair or direct damage reversal. Moreover, these lesions block DNA and RNA polymerases, resulting in cell death. Here, we describe the chemical synthesis of 5′<i>S</i> and 5′<i>R</i> isomers of 5′,8-cyclodeoxyadenosine triphosphate (cdATP) and demonstrate their ability to be incorporated into DNA by replicative DNA polymerases. DNA synthesis assays revealed that the incorporation of the stereoisomeric cdATPs strongly inhibits DNA polymerase reactions. Surprisingly, the two stereoisomers had different mutagenic profiles, since the <i>S</i> isomer of cdATP could be inserted opposite to the dTMP, but the <i>R</i> isomer of cdATP could be inserted opposite to the dCMP. Kinetic analysis revealed that the <i>S</i> isomer of cdATP could be incorporated more efficiently (25.6 μM^–1 min^–1) than the <i>R</i> isomer (1.13 μM^–1 min^–1) during DNA synthesis. Previous data showed that the <i>S</i> isomer in DNA blocked DNA synthesis and the exonuclease activity of DNA polymerase and is less efficiently repaired by NER. This indicates that the <i>S</i> isomer has a tendency to accumulate on the genome DNA, and as such, the <i>S</i> isomer of cdATP may be a candidate cytotoxic drug

Structural Changes of the Active Center during the Photoactivation of <i>Xenopus</i> (6–4) Photolyase

Photolyases (PHRs) repair the UV-induced photoproducts, cyclobutane pyrimidine dimer (CPD) or pyrimidine-pyrimidone (6–4) photoproduct [(6–4) PP], restoring normal bases to maintain genetic integrity. CPD and (6–4) PP are repaired by substrate-specific PHRs, CPD PHR and (6–4) PHR, respectively. Flavin adenine dinucleotide (FAD) is the chromophore of both PHRs, and the resting oxidized form (FAD^ox), at least under <i>in vitro</i> purified conditions, is first photoconverted to the neutral semiquinoid radical (FADH^•) form, followed by photoconversion into the enzymatically active fully reduced (FADH^–) form. Previously, we reported light-induced difference Fourier transform infrared (FTIR) spectra corresponding to the photoactivation process of <i>Xenopus</i> (6–4) PHR. Spectral differences between the absence and presence of (6–4) PP were observed in the photoactivation process. To identify the FTIR signals where these differences appeared, we compared the FTIR spectra of photoactivation (i) in the presence and absence of (6–4) PP, (ii) of ¹³C labeling, ¹⁵N labeling, and [¹⁴N]­His/¹⁵N labeling, and (iii) of H354A and H358A mutants. We successfully assigned the vibrational bands for (6–4) PP, the α-helix and neutral His residue(s). In particular, we assigned three bands to the CO groups of (6–4) PP in the three different redox states of FAD. Furthermore, the changed hydrogen bonding environments of CO groups of (6–4) PP suggested restructuring of the binding pocket of the DNA lesion in the process of photoactivation