Product Analysis of GG-Specific Photooxidation of DNA via Electron Transfer: 2-Aminoimidazolone as a Major Guanine Oxidation Product

Product Analysis of GG-Specific Photooxidation of DNA via Electron Transfer:  2-Aminoimidazolone as a Major Guanine Oxidation Produc

Binding of RARα to the <i>Csn3</i> promoter region containing the DR5 RARE motif.

<p>(<b>A</b>) Schematic illustration of the mouse <i>Csn3</i> promoter region. The <i>Csn3</i> DR5 RARE (filled box) and exon 1 (open box) are shown. Arrows under the nucleotide indicate the positions of the PCR primers used for chromatin immunoprecipitation (ChIP) assays. The numbers indicate position relative to the transcriptional start site (+1). (<b>B</b>) ChIP assays of RARα in <i>Csn3</i> promoter region in P19 cells treated with ATRA. DNA sequences within chromatin that had been immunoprecipitated with anti-RARα antibody (lane 2) or non-specific mouse IgG (negative control, lane 3) were amplified using primer pairs for the region including the DR5 RARE in <i>Csn3</i> promoter (−206 to +1) or the distal region from the target site (−739 to −575). Aliquots of the chromatin before immunoprecipitation were used as a positive control (Input, lane 1). Lane 4 contains non-template DNA.</p

Nucleotide Excision Repair of 5-Formyluracil in Vitro Is Enhanced by the Presence of Mismatched Bases^†

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. Although this residue is a potentially mutagenic lesion and is removed by several base excision repair enzymes, it is unknown whether fU is the substrate of nucleotide excision repair (NER). Here, we analyzed the binding specificity of XPC−HR23B, which initiates NER, and cell-free NER activity on fU opposite four different bases. The result of the gel mobility shift assay showed that XPC−HR23B binds the fU-containing substrates in the following order:  fU:C ≫ fU:T > fU:G > fU:A. Furthermore, in the presence of XPC−HR23B, the dual incision activity was the same as the order of the binding affinity of XPC−HR23B to fU. Therefore, it is concluded that even fU, regarded as a shape mimic of thymine, can be recognized as a substrate of NER incision, and the efficiency depends on instability of the base pair

Effects of RAR agonists on <i>Csn3</i> expression in P19 cells.

<p>(<b>A</b>) RT-PCR analysis of the expression of <i>Csn3</i> and genes encoding the RAR subtypes (<i>Rara</i>, <i>Rarb</i>, and <i>Rarg</i>) during the neural differentiation in P19 cells. Total RNA was isolated from P19 cells at the time indicated following ATRA treatment, and used for cDNA synthesis. PCR analysis was performed with primer sets specific for <i>Csn3</i>, each <i>Rar</i> or for <i>Gapdh</i>. PCR products were then subjected to electrophoresis through a 1.5% agarose gel and subsequently stained with ethidium bromide. This experiment was repeated three times with similar results, and representative pictures are shown here. (<b>B</b>) RT-PCR analysis of <i>Csn3</i>, <i>Rara</i>, <i>Rarb</i>, and <i>Rarg</i> mRNA expression in P19 cells treated with RAR agonists. Total RNA was extracted from P19 cells treated with 100 nM Am80 (RARα agonist; upper panel) or 100 nM AC-41848 (RARγ agonist; lower panel), and <i>Csn3</i> and genes encoding the RAR subtypes expressions were evaluated by RT-PCR analysis. <i>Gapdh</i> was used as a loading control. Numbers in parentheses next to the gene symbols indicate the number of PCR cycles. RT-PCR experiments were repeated at least three times with similar results.</p

Identification of transcriptional regulatory region of the mouse <i>Csn3</i>.

<p>(<b>A</b>) Transcriptional regulatory activity of the <i>Csn3</i> proximal promoter in P19 cells. Schematic representations of the <i>Csn3</i> promoter 5′-deletion constructs used for transient transfections are shown on the left. P19 cells were transfected with the indicated deletion constructs and then treated with ATRA for 48 h followed by luciferase activity determination. Luciferase activity (right) was normalized against <i>Renilla</i> luciferase activity derived from cotransfected pGL4.74[<i>hRluc</i>/TK] reporter vector. Values represent the mean plus S.D. from three independent experiments. Student’s <i>t</i>-test was used to assess statistical significance. Asterisk indicates <i>P</i><0.01 compared with value of construct containing the region between −500 to +39. (<b>B</b>) Map of the mouse <i>Csn3</i> promoter region. The putative DR5 RARE (filled box) and exon 1 (open box) are shown. The numbers indicate the positions relative to the transcriptional start site (+1). Arrows under the sequence indicate the location and orientation of the potential half sites for RAR/RXR binding of putative DR5 RARE (−152/−136). (<b>C</b>) Identification of functional sites within the mouse <i>Csn3</i> promoter region. P19 cells were transfected with wild-type (WT), deleted (DR5d), or mutated (DR5m) RARE-<i>luc</i> vector and treated with ATRA for 48 h. Schematic representations of the constructs and nucleotide sequence used for transfection are shown on the left and bottom panel, respectively. The mutated bases are indicated in italics. Luciferase activity (right) was normalized to <i>Renilla</i> luciferase activity. Values represent the mean plus S.D. from three independent experiments. Student’s <i>t</i>-test was used to assess statistical significance. Asterisk indicates <i>P</i><0.01 compared with value of WT DR5 RARE-<i>luc</i> vector.</p

Expression of <i>Csn3</i> in mouse ES cells and in mouse embryonic development.

<p>(<b>A</b>) Time course RT-PCR analysis of <i>Csn3</i> mRNA expression in mouse ES cells (EB5). Total RNA was extracted from EB5 cells treated with ATRA (upper panel) or DMSO vehicle (lower panel) after 0, 3, 6, 12, 24, 48, 72 or 96 h. RT-PCR was performed using gene-specific primers as described in Materials and Methods. Expression of <i>Oct3/4</i> and <i>Mash1</i> were also examined to show the differentiating status. <i>Gapdh</i> was used as a loading control. PCR products were then subjected to electrophoresis through a 1.5% agarose gel and stained with ethidium bromide. Numbers in parentheses next to the gene symbols indicate the number of PCR cycles. (<b>B</b>) RT-PCR analysis of <i>Csn3</i> mRNA expression in mouse embryos at different developmental stages (E7–E17). The amplified PCR products from the mouse embryos were resolved in 1.5% agarose gel and stained with ethidium bromide. <i>Gapdh</i> was used as a loading control.</p

Molecular interaction between RARα and the <i>Csn3</i> promoter sequence containing the DR5 RARE motif.

<p>(<b>A</b>) Electrophoretic gel mobility shift assays (EMSAs) of the mouse <i>Csn3</i> DR5 RARE. EMSA was performed following incubation of Alexa680-labeled, double-stranded <i>Csn3</i> DR5 RARE probe corresponding to −160/−131 of <i>Csn3</i> promoter (5′-ACTAAGACTGACCTGCAGGTGACCCTGGTG-3′) with nuclear extract from P19 cells that had been treated with ATRA for 3 h (lane 2) or with no added protein (lane 1). For competition assay, unlabeled homologous oligonucleotides were added in increasing amounts (5- or 20-fold excess) to the binding reactions; the unlabeled oligonucleotides functioned as competitors with the labeled probe (lane 3 and 4, respectively). NS represents the non-specific interactions. (<b>B</b>) EMSA for assaying RARα binding to <i>Csn3</i> DR5 RARE. Alexa680-labeled <i>Csn3</i> DR5 RARE probe was incubated with no added protein (lane 1) or with nuclear extract from P19 cells that had been treated with ATRA for 3 h (lane 2). Supershift assay was performed by addition of anti-RARα antibody (lane 3). The arrowhead indicates the supershifted band.</p

ATRA-dependent induction of <i>Csn3</i> gene expression in P19 cells.

<p>(<b>A</b>) Time course RT-PCR analysis of <i>Csn3</i> mRNA expression in P19 cells. Total RNA was extracted from P19 cells treated with ATRA (upper panel) or DMSO vehicle (lower panel) after 0, 3, 6, 12, 24, 36, or 48 h. RT-PCR was performed using gene-specific primers as described in Materials and Methods. Expression of pluripotency marker (<i>Oct3/4</i>) and neurogenic bHLH gene (<i>Mash1</i>, <i>Neurog1</i> and <i>NeuroD1</i>) were also examined to show the differentiating status. Glyceraldehyde-3-phosphate-dehydrogenase (<i>Gapdh</i>) was used as a loading control. PCR products were then subjected to electrophoresis through a 1.5% agarose gel and stained with ethidium bromide. Numbers in parentheses next to the gene symbols indicate the number of PCR cycles. RT-PCR experiments were repeated at least three times with similar results, and representative pictures are shown here. (<b>B</b>) Real-time PCR analysis of <i>Csn3</i> mRNA expression in P19 cells following ATRA treatment. Total RNA extracted from P19 cells that had been treated with ATRA for 0, 3, 6, 12, 24, or 48 h was used as template in this analysis. Real-time PCR was performed to assess <i>Csn3</i> expression; and <i>Csn3</i> expression was normalized relative to expression of the housekeeping gene hydroxymethylbilane synthase (<i>Hmbs</i>). All data points represent the mean plus S.E. from three independent experiments.</p

Analysis of Nucleotide Insertion Opposite 2,2,4-Triamino-5(2<i>H</i>)‑oxazolone by Eukaryotic B- and Y‑Family DNA Polymerases

Mutations induced by oxidative DNA damage can cause diseases such as cancer. In particular, G:C–T:A and G:C–C:G transversions are caused by oxidized guanine and have been observed in the <i>p53</i> and <i>K-ras</i> genes. We focused on an oxidized form of guanine, 2,2,4-triamino-5­(2<i>H</i>)-oxazolone (Oz), as a cause of G:C–C:G transversions based on our earlier elucidation that DNA polymerases (Pols) α, β, γ, ε, η, I, and IV incorporate dGTP opposite Oz. The nucleotide insertion and extension of Pols δ, ζ, ι, κ, and REV1, belonging to the B- and Y-families of DNA polymerases, were analyzed for the first time. Pol δ incorporated dGTP, in common with other replicative DNA polymerases. Pol ζ incorporated dGTP and dATP, and the efficiency of elongation up to full-length beyond Oz was almost the same as that beyond G. Although nucleotide incorporation by Pols ι or κ was also error-prone, they did not extend the primer. On the other hand, the polymerase REV1 predominantly incorporated dCTP opposite Oz more efficiently than opposite 8-oxo-7,8-dihydroguanine, guanidinohydantoin, or tetrahydrofuran. Here, we demonstrate that Pol ζ can efficiently replicate DNA containing Oz and that REV1 can prevent G:C–C:G transversions caused by Oz

Additional file 1 of Analysis of nucleotide insertion opposite urea and translesion synthesis across urea by DNA polymerases

Additional file 1: Fig. S1. Construction of the 30-merUa. Two products (30-merUa1 and 30-merUa2) were obtained (Materials and methods). However, these two products equilibrate with each other and thus could not be isolated separately. In a previous report [7], Dubey, et al. revealed that Ua comprises the α- and β-anomers. The mixture of the two products (C289H370N103O176P29) was confirmed by ESI-MS (m/z 9000.727) and then was used as 30-merUa in our experiment