HERPES-SIMPLEX VIRUS-DNA REPLICATION - A SPACER SEQUENCE DIRECTS THE ATP-DEPENDENT FORMATION OF A NUCLEOPROTEIN COMPLEX AT ORI(S)

The origin-binding protein (OBP) from herpes simplex virus 1 is a member of the SF2 helicase superfamily and is required for the initiation of DNA synthesis from a viral origin of DNA replication (ori(s)). The high-affinity binding sites for OBP in ori(s), boxes I and II, are separated by an A+T-rich spacer. We used the gel retardation technique to examine the influence of this spacer sequence on the formation of a specific complex, referred to as complex II, between OBP and ori(s). The formation of this OBP-ori(s) complex was greatly promoted by adenosine 5'-[gamma-thio]triphosphate and other nucleotide cofactors. Surprisingly, ori(s) constructs where the spacer sequence had been altered with approximately half of a helical turn (+4 or -6 base pairs) supported the formation of a more stable complex II than the wild-type origin. DNase I footprinting experiments showed that the cooperative binding of OBP to boxes I and II was affected by the length of the spacer sequence in the same way. In contrast, the ability of ori(s)-containing plasmids to replicate was most efficient with wild-type ori(s). This paradox can be resolved if it is assumed that an ATP-dependent cooperative binding of OBP to properly spaced recognition sequences in ori(s) is required to induce a conformational change of DNA, thereby facilitating initiation of DNA replication.