DNA replication catalyzed by Herpes Simplex Virus Type 1 proteins reveals trombone loops at the fork [Molecular Biophysics]

December 3rd, 2014 by Bermek, O., Willcox, S., Griffith, J. D.

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 >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 ss 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. Further, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis.