title: DNA polymerase θ specializes in incorporating synthetic expanded-size (xDNA) nucleotides.
abstract: DNA polymerase θ (Polθ) is a unique A-family polymerase that is essential for alternative end-joining (alt-EJ) of double-strand breaks (DSBs) and performs translesion synthesis. Because Polθ is highly expressed in cancer cells, confers resistance to ionizing radiation and chemotherapy agents, and promotes the survival of homologous recombination (HR) deficient cells, it represents a promising new cancer drug target. As a result, identifying substrates that are selective for this enzyme is a priority. Here, we demonstrate that Polθ efficiently and selectively incorporates into DNA large benzo-expanded nucleotide analogs (dxAMP, dxGMP, dxTMP, dxAMP) which exhibit canonical base-pairing and enhanced base stacking. In contrast, functionally related Y-family translesion polymerases exhibit a severely reduced ability to incorporate dxNMPs, and all other human polymerases tested from the X, B and A families fail to incorporate them under the same conditions as Polθ. We further find that Polθ is inhibited after multiple dxGMP incorporation events, and that Polθ efficiency for dxGMP incorporation approaches that of native dGMP. These data demonstrate a unique function for Polθ in incorporating synthetic large-sized nucleotides and suggest the future possibility of the use of dxG nucleoside or related prodrug analogs as selective inhibitors of Polθ activity.
authors: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA.
authors: Tatiana
authors: T
authors: Kent
authors: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA.
authors: Timur D
authors: TD
authors: Rusanov
authors: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA.
authors: Trung M
authors: TM
authors: Hoang
authors: Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
authors: Willem A
authors: WA
authors: Velema
authors: Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
authors: Andrew T
authors: AT
authors: Krueger
authors: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors: William C
authors: WC
authors: Copeland
authors: Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
authors: Eric T
authors: ET
authors: Kool
authors: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA richard.pomerantz@temple.edu.
authors: Richard T
authors: RT
authors: Pomerantz
mesh_terms: D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D000097462:DNA Polymerase theta
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase; D009711:Nucleotides; D000097462:DNA Polymerase theta