Leading and Lagging Strand DNA Synthesis in vitro by a Reconstituted Herpes Simplex Virus Type 1 Replisome

• Published in: Proceedings of the National Academy of Sciences - PNAS 2000-04, Vol.97 (8), p.3896-3900
• Main Authors: Falkenberg, Maria, Lehman, I. R., Elias, Per
• Format: Article
• Language: English
• Subjects: Biochemistry; Biological Sciences; Circles; Deoxyribonucleic acid; DNA; DNA Helicases; DNA Helicases - metabolism; DNA Primase; DNA Replication; DNA Replication - genetics; DNA-Binding Proteins; Electrophoresis; Enzymes; Gels; Genetic; genetics; Herpes simplex virus 1; Herpes viruses; Herpesviridae; Human herpesvirus 1; ICP8 protein; Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy); Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci); metabolism; Microbiology in the medical area; Mikrobiologi inom det medicinska området; Phage T7; Protein Binding; Proteins; replisomes; ribonucleoside triphosphate; ribonucleoside triphosphates; Ribonucleosides; Simplexvirus; Simplexvirus - genetics; Simplexvirus - metabolism; Templates; Templates, Genetic; UL42 protein; Viral Proteins; Viral Proteins - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.8.3896

The synthesis of double-stranded DNA by a rolling circle mechanism was reconstituted in vitro with a replisome consisting of the DNA polymerase-UL42 complex and the heterotrimeric helicaseprimase encoded by herpes simplex virus type 1. Okazaki fragments 3 kilobases in length and leading strands that may exceed 10 kilobases are produced. Lagging strand synthesis is stimulated by ribonucleoside triphosphates. DNA replication appears to be processive because it resists competition with an excess of (dT)150/(dA)20. The single-strand DNA binding protein ICP8 is not required, and high concentrations of ICP8 can, in fact, inhibit lagging strand synthesis. The inhibition can, however, be overcome by the addition of an excess of the UL8 component of the helicase-primase. Rolling circle replication by the herpesvirus and bacteriophage T7 replisomes appears to proceed by a similar mechanism.