Activation of Herpes simplex virus type 1 origin of DNA replication

Abstract: Herpes simplex virus type 1 has been used to study the molecular mechanism of DNA replication. Initiation of DNA replication takes place at specific regions called origins of replication. The HSV-1 genome contains three origins of replication, one copy of oriL and two copies of oriS. They contain palindromes of 144 and 46 base pairs with a central AT rich region. Two high affinity binding sites for the viral origin binding protein, OBP, surround the AT rich spacer sequence. In oriS, the two binding sites are referred to as box I and box II. An additional putative binding site referred to as box III has much lower binding affinity. DNA replication requires activation of origins by the origin binding protein, OBP. OBP is the product of UL9 gene and it is one of seven proteins required for viral DNA replication. It is an origin specific DNA binding protein and an ATP dependent DNA helicase. We have focused our interest on the interaction between OBP and oriS, and we have examined functional and structural properties crucial for initiation of DNA replication. We have found that during initiation of HSV-1 DNA replication oriS is converted to a novel conformation referred to as oriS'. This new conformation is stably bound by OBP.We have examined the structural features of oriS' and found that oriS' is composed of a hairpin formed by complementary intra-strand base pairing between boxI and box III. It also contains a 3´ tail composed of the AT rich region. The box I/box III hairpin is evolutionarily conserved in several alpha herpes viruses. We also showed that oriS' is an efficient activator of the OBP dependent ATPase.Finally, we have identified conditions for unwinding of duplex oriS and formation of oriS' catalyzed by OBP. We have showed that conversion of oriS to oriS' by OBP is dependent on interaction of OBP with the HSV-1 single strand DNA binding protein ICP8 and hydrolysable ATP. We suggest that activation of duplex oriS proceeds from sequence specific binding of OBP to oriS towards to the formation of a stable intermediate composed of the OBP/oriS' complex. This complex may serve as an assembly site for the herpes virus replisome.