A novel conformation of the Herpes simplex virus origin of DNA replicationrecognized by the origin binding protein

The Herpes simplex virus type I origin binding protein (OBP) is a sequence- specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene. It is required for the activation of the viral origin of DNA replicat ion oriS. Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS*. Studies using S1 nuclease suggest that oriS* consists of a cen tral hairpin with an AT-rich sequence in the loop. Single-stranded oligonuc leotides corresponding to the upper strand of oriS can adopt the same struc ture. OBP forms a stable complex with oriS*. We have identified structural features of oriS* recognized by OBP. The central oriS palindrome as well as sequences at the 5' side of the oriS palindrome were required for complex formation. Importantly, we found that mutations that have been shown to red uce oriS-dependent DNA replication also reduce the formation of the OBP-ori S*: complex. We suggest that oriS*: serves as an intermediate in the initia tion of DNA replication providing the initiator protein with structural inf ormation for a selective and efficient assembly of the viral replication ma chinery.