title: DNA replication catalyzed by herpes simplex virus type 1 proteins reveals trombone loops at the fork.
abstract: Using purified replication factors encoded by herpes simplex virus type 1 and a 70-base minicircle template, we obtained robust DNA synthesis with leading strand products of >20,000 nucleotides and lagging strand fragments from 600 to 9,000 nucleotides as seen by alkaline gel electrophoresis. ICP8 was crucial for the synthesis on both strands. Visualization of the deproteinized products using electron microscopy revealed long, linear dsDNAs, and in 87%, one end, presumably the end with the 70-base circle, was single-stranded. The remaining 13% had multiple single-stranded segments separated by dsDNA segments 500 to 1,000 nucleotides in length located at one end. These features are diagnostic of the trombone mechanism of replication. Indeed, when the products were examined with the replication proteins bound, a dsDNA loop was frequently associated with the replication complex located at one end of the replicated DNA. Furthermore, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis.
authors.affiliation: From the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295.
authors.forename: Oya
authors.initials: O
authors.lastname: Bermek
authors.affiliation: From the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295.
authors.forename: Smaranda
authors.initials: S
authors.lastname: Willcox
authors.affiliation: From the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295 jdg@med.unc.edu.
authors.forename: Jack D
authors.initials: JD
authors.lastname: Griffith
mesh_terms: D004261:DNA Replication / Q000235:genetics* / Q000502:physiology; D004279:DNA, Viral / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D014764:Viral Proteins
keywords: DNA Replication; Electron Microscopy (EM); Gel Electrophoresis; Herpesvirus; Structural Biology; Virology