Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.
The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Gudrun
G
Stengel
Robert D
RD
Kuchta
D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D013698:Templates, Genetic; D013938:Thymidine Monophosphate / Q000378:metabolism; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D004270:DNA, Circular; D004279:DNA, Viral; D003838:Deoxyadenine Nucleotides; D014764:Viral Proteins; D013938:Thymidine Monophosphate; C012873:2'-deoxy-5'-adenosine monophosphate