Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome - PubMed (original) (raw)

Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome

M Falkenberg et al. Proc Natl Acad Sci U S A. 2000.

Abstract

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 helicase-primase 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.

PubMed Disclaimer

Figures

Figure 1

Minicircle template for rolling circle DNA replication. The minicircle template was prepared as described in Materials and Methods. [α-32P]dCTP was used to preferentially label the leading strand, and [α-32P]dGTP was used to label the lagging strand fragments. The single G and C in the templates for lagging and leading strands are indicated with an asterisk.

Figure 2

Products of leading and lagging strand synthesis. Products formed by rolling circle DNA replication in vitro were analyzed by alkaline agarose gel electrophoresis and restriction enzyme cleavage as described in Materials and Methods. The reaction mixtures contained 50 fmol of DNA polymerase-UL42, 350 fmol of the UL5/UL52/UL8 heterotrimer, 500 fmol of ICP8, and 35 fmol of the minicircle template. (A) Autoradiograph of an alkaline agarose gel. First lane, DNA size markers; second lane, products labeled by [α-32P]dGTP; third lane, products labeled by [α-32P]dCTP. (B) Autoradiograph of Southern blot analyses of replication products. First lane, the oligonucleotide probe is complementary to the lagging strand; second lane, the oligonucleotide probe is complementary to the leading strand; third lane, DNA size markers. (C) Products labeled with [α-32P]dCTP and [α-32P]dGTP were cleaved with _Mbo_I and were subjected to polyacrylamide gel electrophoresis as described in Materials and Methods.

Figure 3

Lagging strand synthesis is stimulated by ribonucleoside triphosphates. The reaction mixtures were prepared as described in Materials and Methods. ICP8 and the ribonucleoside triphosphates GTP, CTP, and UTP were omitted as indicated. (A) Products were analyzed by alkaline agarose gel electrophoresis. (B) The products were cleaved with _Mbo_I and were analyzed by PAGE.

Figure 4

Functional interaction between UL8 protein and ICP8. (A) The reaction mixture contained [α-32P]dGTP 350 fmol of the UL5/52 subassembly and 50 fmol of DNA polymerase-UL42. 700 fmol UL8 and 0.5 or 5 pmol ICP8 were added as indicated. _Mbo_I cleavage and PAGE was performed as described in Materials and Methods. (B) The reaction mixture contained [α-32P]dGTP, 350 fmol of the UL5/52 subassembly, and 50 fmol of DNA polymerase-UL42. Increasing amounts of ICP8 were added as indicated. The samples were processed as above. (C) The reaction mixture contained [α-32P]dGTP, 350 fmol of the UL5/52 subassembly, 50 fmol of DNA polymerase-UL42, and 1 pmol of ICP8. Increasing amounts of UL8 protein were added as indicated. The molar ratio of UL5/52 to UL8 is shown.

Figure 5

Rolling circle replication by the HSV-1 replisome is resistant to challenge by (dT)150/(dA)20. Reaction conditions were as described in Materials and Methods. (dT)150/(dA)20was added to a final concentration of 0.32 μM at 0, 5, and 15 min as indicated by the arrows. Samples were processed by restriction enzyme cleavage and PAGE as described in Materials and Methods. No competitor DNA was added to the experiment shown in the first six lanes.

References

1. McGeoch D J, Davison A J. In: Origin and Evolution of Viruses. Domingo E, Webster R, Holland J, editors. New York: Academic; 1999. pp. 441–465.
1. Roizman B, Sears A E. In: Fields Virology. Fields B N, Knipe D M, Howley P M, editors. Philadelphia: Lippincott; 1996. pp. 2297–2342.
1. Challberg M D. Proc Natl Acad Sci USA. 1986;83:9094–9098. - PMC - PubMed
1. Lehman I R, Boehmer P E. J Biol Chem. 1999;274:28059–28062. - PubMed
1. Alberts B M, Morris C, Mace D, Sinha N, Bittner M, Moran L. In: DNA Synthesis and Its Regulation. Goulian M, Hanawalt P, Fox C F, editors. Menlo Park, CA: Benjamin; 1975. pp. 241–269.

Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome - PubMed (original) (raw)