STUDY OF THE STRUCTURE OF REPLICATIVE INTERMEDIATES OF HSV-1 DNA BY PULSED-FIELD GEL-ELECTROPHORESIS

DNA from HSV-1-infected cells was separated by pulsed-field gel electr ophoresis into two virus-specific bands: one that migrated as the line ar monomer genome (152 kb) and another that remained at the origin of the gel. The latter band contained the replicating HSV-1 DNA, as deter mined by pulse-labeling with H-3thymidine. To investigate the struct ure of this ''gel origin'' DNA, we constructed a HSV-1 KOS mutant bear ing a unique PacI restriction site (HSV-1 PAC1DTK). Partial digestion of gel origin PAC1DTK DNA at late times postinfection (24-48 hr) demon strated the presence of linear concatemers on pulsed-field gel electro phoresis. Within each concatemer, the long (L) regions of adjacent mon omer genomes were found in the two possible orientations. In addition, shorter-than-unit-size fragments that corresponded in size to the lef t end fragments of the viral genome were detected with the U-L region in the two possible orientations. At early times postinfection (8-12 h r), digestion with PacI released only a trace of linear fragments, and most of the gel origin DNA did not migrate on pulsed-field gel electr ophoresis. Multiple cuts with EcoRI (a restriction enzyme that cuts th e HSV-1 KOS genome 12 times) were necessary to release linear fragment s that migrated from the origin of the gel. These results indicate tha t replicative intermediates of HSV-1 DNA are linked in a large network that needs to be unraveled before packaging takes place. This network may be composed of linear molecules linked together by frequent recom bination events or of products of a mode of replication other than sim ple rolling circle (e.g., theta replication). (C) 1994 Academic Press, Inc.