Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • br Experimental Procedures DNA synthesis reagents

    2020-11-20


    Experimental Procedures DNA synthesis reagents were purchased from Glen Research (Sterling, VA). A solution of 0.1 M triethylamine acetate (TEAA) (pH 6.5) was used as HPLC buffer A, and HPLC-grade acetonitrile from Oceanpak (Sweden) was used as HPLC buffer B. All other chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Milli-Q water (resistance > 18 MΩ cm) was used to prepare all solutions. The general methodology about the characterization of MDM, simulation of lipid residue, calculation of DNA amount, calculation of standard electrode potential, cell culture, cytotoxicity study, internalization study of MDM using flow cytometer, and confocal laser-scanning microscopy imaging are described in the Supplemental Experimental Procedures.
    Acknowledgments This work is supported by the National Institutes of Health (GM 127130), the National Science Foundation (NSF1645215), and the National Natural Science Foundation of China (21521063). We thank Mr. Yongwei Wang for his inspirational comments about this paper.
    Introduction DNA polymerases (DNAPs) are key enzymes essential for genome replication, recombination, and repair across all cellular life forms and their viruses. Family B DNAPs (PolBs) are involved in genome replication in Eukarya and Archaea, but also in viruses from the three domains of life (Koonin, 2006). For the initiation of DNA synthesis, all PolBs characterized thus far depend on the presence of an external primer, a hydroxyl group presented either by a nucleic 3-AP (RNA or DNA) or the so-called terminal protein (TP). Thus, based on the primer requirement for the initiation of genome replication as well as phylogenetic clustering, these enzymes can be broadly divided into two major groups (Filée et al., 2002): RNA primed (rPolBs) and protein primed (pPolBs). The evolutionary relationship between the two groups is unknown, and it is thus unclear whether the ancestral PolB would have employed a protein or an RNA oligonucleotide as a primer. The rPolB group contains mainly replicases devoted to accurate and efficient copying of large cellular and viral genomes. By contrast, pPolBs are exclusive to selfish mobile genetic elements (MGEs) and viruses with moderately sized linear genomes (<50 kb) (Kazlauskas and Venclovas, 2011, Krupovic and Koonin, 2015). The signature of pPolBs is the presence of specific subdomains, named TPR1 and TPR2, which were originally described in Φ29 DNAP. TPR1 is required for the DNAP interaction with the TP, whereas TPR2 endows pPolB with the processivity and strand displacement capacity (Dufour et al., 2000, Rodríguez et al., 2005, Salas et al., 2016), the two properties ensuring the superiority of Φ29DNAP in various molecular biology applications, such as multiple-displacement single-molecule DNA amplification (Hutchison et al., 2005, Sidore et al., 2016). Protein priming has been described for a number of viruses (Berjón-Otero et al., 2016, Hoeben and Uil, 2013, Salas, 1991) as well as for linear eukaryotic plasmids (Klassen and Meinhardt, 2007). More recently, pPolB-encoding genes were also identified in two superfamilies of MGE integrated into various cellular genomes (Krupovic and Koonin, 2016). The first superfamily comprises eukaryotic virus-like transposable elements, called Polintons (also known as Mavericks), which, besides pPolB, encode retrovirus-like integrases and a set of proteins predicted to be involved in the formation of viral particles (Krupovic et al., 2014a). The second supergroup, denoted casposons, is present in a wide range of archaea and some bacteria (Krupovic et al., 2014b). For integration into the cellular genome, casposons employ endonucleases homologous to Cas1, a signature enzyme of the CRISPR-Cas systems (Béguin et al., 2016, Krupovic et al., 2017). It has been postulated that pPolBs participate in the replication of the casposon and polinton genomes (Kapitonov and Jurka, 2006, Krupovic et al., 2014b); accordingly, these MGEs are referred to as self-synthesizing or self-replicating elements.