A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation

<div><p>Several germline single nucleotide polymorphisms (SNPs) have been identified in the <em>POLB</em> gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β), encoded by the <em>POLB</em> gene, is the main gap-filling polymerase involved in base excision repair (BER), a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the <em>POLB</em> germline coding SNP (rs3136797) in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility.</p> </div

Chromosomal aberrations in P242R-expressing cells.

<p>Representative metaphase spread of MCF10A expressing (A.) WT or (B.) P242R Pol β. Chromosomal fusions are shown with the gray arrow and fragments are shown with black arrows. C. Number of aberrations per metaphase. A total of at least 50 metaphases were scored for each cell line.</p

The P242R germline variant of Pol β is slow and binds DNA tightly.

<p>A. Representative results from a presteady-state burst assay. Results for the WT are shown as filled squares fit with a solid curve. Results for the P242R are shown as open triangles fit with a dashed curve. The assay was repeated four times for each protein. B. Representative results from a gel electrophoretic mobility shift assay. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003052#s2" target="_blank">Results</a> for the different proteins are shown as in A.</p

Accumulation of BER intermediates in MCF10A cells expressing P242R Pol β.

<p>A. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 30 minutes and allowed to recover for 0, 30, or 60 min and single-strand breaks (SSBs) were analyzed by comet assay. The percentage of tail DNA is plotted on the Y-axis. B. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 30 min and allowed to recover for 0, 30, or 60 min, stained with γH2AX antibody, and analyzed by flow cytometry. Cells were treated for 120 min as a positive control. C–D. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 2 h and allowed to recover for 0 or 2 hours. Cells were stained with γH2AX antibody and propidium iodide to assess the levels of double-strand breaks (DSBs) and the cell cycle phase, respectively, and analyzed by flow cytometry. Data are plotted as the mean ± SEM. Data are plotted as the mean ± SEM (n = 3). A and C. ** and *** denote <i>p</i><0.01 and 0.001, respectively. ∧ denotes <i>p</i><0.05 comparing 0 vs 2 h recovery within each cell line. ∧∧∧ denotes <i>p</i><0.001 comparing 30 or 60 min recovery to 0 recovery. D. *** denotes <i>p</i><0.001 comparing WT+MMS to P242R+MMS in each phase of the cell cycle. ∧ and ∧∧∧ denote <i>p</i><0.05 and 0.001, respectively, comparing WT+MMS+recovery to P242R+MMS+recovery in each phase of the cell cycle.</p

P242R Pol β confers slight sensitivity to MMS compared to WT.

<p>Clonogenic survival assays were conducted with (A.) Pol β^−/− MEFs, (B.) Pol β^+/+ MEFs, or (C.) MCF10A pools expressing WT or P242R Pol β. Filled circles represent results from pools expressing empty vector, filled squares represent pools expressing WT Pol β, and filled triangles represent pools expressing P242R Pol β. Data are plotted as the mean ± SEM (n = 3).</p

Site-specific Genome Editing in PBMCs With PLGA Nanoparticle-delivered PNAs Confers HIV-1 Resistance in Humanized Mice.

Biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating triplex-forming peptide nucleic acids (PNAs) and donor DNAs for recombination-mediated editing of the CCR5 gene were synthesized for delivery into human peripheral blood mononuclear cells (PBMCs). NPs containing the CCR5-targeting molecules efficiently entered PBMCs with low cytotoxicity. Deep sequencing revealed that a single treatment with the formulation resulted in a targeting frequency of 0.97% in the CCR5 gene and a low off-target frequency of 0.004% in the CCR2 gene, a 216-fold difference. NP-treated PBMCs efficiently engrafted immunodeficient NOD-scid IL-2rγ(-/-) mice, and the targeted CCR5 modification was detected in splenic lymphocytes 4 weeks posttransplantation. After infection with an R5-tropic strain of HIV-1, humanized mice with CCR5-NP-treated PBMCs displayed significantly higher levels of CD4(+) T cells and significantly reduced plasma viral RNA loads compared with control mice engrafted with mock-treated PBMCs. This work demonstrates the feasibility of PLGA-NP-encapsulated PNA-based gene-editing molecules for the targeted modification of CCR5 in human PBMCs as a platform for conferring HIV-1 resistance.Molecular Therapy-Nucleic Acids (2013) 2, e135; doi:10.1038/mtna.2013.59; published online 19 November 2013. Mol Ther Nucleic Acids 2013 Nov 19; 2:e135