Herpes simplex virus DNA synthesis at a preformed replication fork in vitro.
Proteins from herpes simplex virus (HSV)-infected cells were used to reconstitute DNA synthesis in vitro on a preformed replication fork. The preformed replication fork consisted of a nicked, double-stranded, circular DNA molecule with a 5' single-strand tail that was noncomplementary to the template. The products of DNA synthesis on this substrate were rolling-circle molecules, as demonstrated by electron microscopy and alkaline agarose gel electrophoresis. The tails contained double-stranded regions, indicating that both leading- and lagging-strand DNA syntheses occurred. Rolling-circle DNA replication was dependent upon HSV DNA polymerase and ATP and was stimulated by a crude fraction containing ICP8 (HSV DNA-binding protein). Similar protein fractions from mock-infected cells were unable to support rolling-circle DNA replication. This in vitro DNA replication system should prove useful in the identification and characterization of the enzymatic activities required at the HSV replication fork.
Rabkin
S D
SD
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Hanlon
B
B
D000212:Acyclovir / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002472:Cell Transformation, Viral; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000096:biosynthesis / Q000302:isolation & purification* / Q000648:ultrastructure; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D015345:Oligonucleotide Probes; D018139:Simplexvirus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D014709:Vero Cells
D000998:Antiviral Agents; D004279:DNA, Viral; D015345:Oligonucleotide Probes; C025114:acyclovir triphosphate; D004259:DNA-Directed DNA Polymerase; D000212:Acyclovir