Model depicting the structure of Polß.

<p>(A) Structure (pdb2fms) depicting DNA Polymerase ß (Polß) with a gapped DNA substrate and dUMPNPP with magnesium in the catalytic site <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003086#pgen.1003086-Batra1" target="_blank">[35]</a>. The image is a cartoon rendition of the polypeptide chain of Polß in teal, the gapped DNA substrate in salmon, and the incoming dUMPNPP base in green. Amino acids known to be altered by germline mutations are shown using a space-filling rendering: R137 (magenta) and P242 (orange). The fingers, palm, and thumb domains of Polß are indicated. The 8K domain is at the back of the structure, facing away from the plane of the image, and is shown behind the DNA in this orientation. (B) Structure (pdb3lqc) depicting oxidized XRCC1 bound to the Polß palm/thumb domains <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003086#pgen.1003086-Cuneo1" target="_blank">[36]</a>. The image is a cartoon rendition of the palm and thumb domains of Polß in blue, with a mesh illustrating the surface of the structure (amino acids 150–335), and a cartoon rendition of the oxidized form of XRCC1 in green, with a mesh illustrating the surface of the structure (amino acids 1–151). Amino acid P242 (orange) is shown using a space-filling rendering. The images were generated using PyMOL (Molecular Graphics System, Version 1.2r3pre; Schrödinger, LLC; <a href="http://pymol.org/" target="_blank">http://pymol.org/</a>).</p

Germline and somatic <i>POLB</i> mutants.^*

<p><i>n.d.</i> = not determined.</p>*<p>See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003086#pgen.1003086-Donigan2" target="_blank">[53]</a> for an extensive list of <i>POLB</i> gene mutations recently identified in colorectal tumors.</p

Camptothecin Efficacy to Poison Top1 Is Altered by Bisphenol A in Mouse Embryonic Fibroblasts

Bisphenol A (BPA) is used heavily in the production of polycarbonate plastics, thermal receipt paper, and epoxies. Ubiquitous exposure to BPA has been linked to obesity, diabetes, and breast and reproductive system cancers. Resistance to chemotherapeutic agents has also been shown in cancer cell models. Here, we investigated BPA’s ability to confer resistance to camptothecin (CPT) in mouse embryonic fibroblasts (MEFs). MEFs are sensitive to CPT; however, co-exposure of BPA with CPT improved cell survival. Co-exposure significantly reduced Top1-DNA adducts, decreasing chromosomal aberrations and DNA strand break formation. This decrease occurs despite BPA treatment increasing the protein levels of Top1. By examining chromatin structure after BPA exposure, we determined that widespread compaction and loss of nuclear volume occurs. Therefore, BPA reduced CPT activity by reducing the accessibility of DNA to Top1, inhibiting DNA adduct formation, the generation of toxic DNA strand breaks, and improving cell survival

Oxidative damage during HIV infection.

<p><i>(A)</i> OGG1 and MYH both recognize 8-oxo-dG damage (shown as <i>G_o</i>). OGG1 repairs 8-oxo-dG to G. Replication of 8-oxo-dG results in an 8-oxo-dG:A mispair, which is recognized by MYH. The MYH glycosylase initiates repair of the mispaired A to C yielding an 8-oxo-dG:C base pair. The product of the MYH repair reaction must still be repaired by OGG1. <i>(B)</i> Wild type cells were infected with an HIV based vector expressing GFP. Cells were treated with increasing concentrations of H₂O₂ immediately prior to infection. Viability in the absence (open diamonds) or presence of HIV (closed diamonds) was measured by trypan blue exclusion. Error bars indicate the standard deviations from three independent experiments.</p

G:C content surrounding HIV integration sites.

<p>The percentage of HIV integration sites compared to the percentage of G:C base pairs within a 5 kb window is shown for wild type cells compared to (<b>A</b>) <i>OGG1</i> null cells, (<b>B</b>) <i>MYH</i> null cells, (<b>C</b>) wild type cells treated with 10 µM H₂O₂, and (<b>D</b>) wild type cells treated with 30 µM H₂O₂. The frequency of G:C base pairs in the mouse genome is 0.41. There is no significant difference between HIV integration sites in untreated wild type cells and <i>OGG1</i> null (p = 0.96), <i>MYH</i> null (p = 0.99), 10 µM H₂O₂ treated (p = 0.67), or 30 µM H₂O₂ treated cells (p = 0.88).</p

Retroviral integration.

<p><i>(A)</i> Viral cDNA is depicted by a thin line and host target DNA is indicated by a thick line. Base pairs in the host target DNA are numbered. The HIV LTR ends are covalently joined to the target DNA 5 base pairs apart. The intervening host DNA denatures yielding an integration intermediate with two 5 base pair gaps. <i>(B)</i> The sequence preference observed at HIV integration sites. The numbering is identical to (A) and the points of joining are indicated by “IN”. Base pairs in green are favored and base pairs in red are disfavored at HIV integration sites.</p

HIV integration near genomic elements.

<p>HIV integration sites were mapped to genomic elements in <i>OGG1</i> null cells, <i>MYH</i> null cells, untreated wild type cells, and wild type cells treated with 10 µM or 30 µM H₂O₂. (<b>A</b>) HIV has a preference for integration to transcription units. (<b>B</b>) HIV shows no preference for integration to promoters. (<b>C</b>) There is no preference for HIV integration within 5 kb of CpG islands.</p

Effects of 8-oxo-dG specific glycosylases or hydrogen peroxide on HIV integration site sequence preference.

<p>Wild type, <i>OGG1</i> null, <i>MYH</i> null, and wild type cells treated with 10 µM or 30 µM H₂O₂, were infected with an HIV based retroviral vector at 0.8 MOI. 10 µM H₂O₂ is less than the 50% lethal dose for the wild type cells and 30 µM H₂O₂ is greater. After 7 days, genomic DNA was purified. HIV integration sites were subcloned, sequenced, and mapped to the mouse genome. The random frequency of G or C in the mouse genome is 0.205 and A or T is 0.295. The differences in observed base frequencies relative to the random frequencies are shown. Base numbering relative to the HIV points of joining is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103164#pone-0103164-g001" target="_blank">Figure 1</a>. Boxes indicate the known HIV integration base preferences for wild type cells. Green, red, and gray highlights indicate a statistically significant difference of >0.10 from random frequency with a p value of <0.005. Green highlights are positive differences and red highlights are negative previously published palindromic prefered bases. Deletion of the <i>OGG1</i> gene leads to a loss of HIV sequence preference at positions −2 and −1. Deletion of the <i>MYH</i> gene also leads to loss of the HIV sequence preference at positions −2 and 7. While treatment of wild type cells with 10 µM H₂O₂ did not dramatically alter the sequence preference at integration sites, treatment with 30 µM H₂O₂ led to the disfavor of C at position −3 and disfavor of G at position 8, highlighted in red.</p

Infection of BER cell lines with HIV, MMLV, and FIV.

<p>Cells were infected with HIV, MMLV, and FIV retroviral vectors expressing GFP following integration. Cells were analyzed at 72 hpi by flow cytometry for GFP expression indicating successful infection. Wild type MEFs were from littermates. <i>(A)</i> Wild type (WT), <i>Myh</i>−/−, and <i>Ogg1</i>−/− cell lines, <i>(B)</i> Wild type (<i>Neil1</i>+/+) and <i>Neil1</i>−/− cell lines, <i>(C)</i> Wild type (<i>Polß</i>+/+) and <i>Polß</i>−/− cell lines. Infections were performed at two MOI in duplicate at least three times. Error bars indicate the standard deviation after normalization.</p

Integration activity of HIV PICs from BER cell lines.

<p>HIV PIC extracts were generated at 6 hpi. Purified human genomic DNA was added to HIV PICs. Integration products and total HIV cDNA were measured by qPCR. The number of integration products divided by the total HIV cDNA indicates the integration activity. Integration activity of PICs from mutant cells is expressed relative to PICs from matched wild type cells. <i>(A)</i> HIV PICs from matched wild type and <i>Ogg1</i>, <i>Neil1</i>, or <i>Polß</i> null cells. <i>(B)</i> Increasing concentrations of purified recombinant human POLß protein were added to PICs from wild type and <i>Polß</i>−/− cells. PICs were generated from three infections. Integration reactions for each PIC preparation were performed in duplicate. Error bars indicate the standard deviation. <i>(C)</i> Silver stained gel of recombinant human POLß protein. Lanes 1 (left) and 7 (right) show the molecular weight markers. Lanes 2–6 correspond to 20, 40, 80, 120 and 200 ng purified POLß, respectively.</p