Initiation of herpes simplex virus DNA replication

Abstract

During virus infection multiple new copies of the virus genome are made by a DNA synthesis machinery. Initiation of DNA replication involves the recognition of origins of DNA replication by an initiator protein. The genome of Herpes simplex virus type-1, HSV-1, contains three largely homologous origins of replication: one copy of oriL and two copies of oriS. The origins of DNA replication are activated by the HSV-1 origin binding protein OBP, which is the product of the UL9 gene. A virus specific replication machinery consisting of six proteins is then assembled at the origins. It consists of a heterodimeric DNA polymerase, a single-strand DNA-binding protein and a heterotrimeric helicase-primase complex.This thesis focuses on interactions between oriS and OBP. We have examined the stoichiometry of complexes formed between the DNA binding domain of OBP and its recognition sequence, GTTCGCAC, and established that a 1:1 complex forms. We have identified phosphates and methyl groups in DNA that are involved in the sequence specific recognition of oriS. The results show that OBP primarily recognizes its target sequence through major groove interactions. Interestingly, we find that single-stranded versions of HSV-1 oriS can adopt a novel conformation referred to as oriS*, which forms a stable and specific complex with OBP. Genetic experiments suggest that oriS* is formed as an intermediate during initiation of HSV-1 DNA replication. On the basis of these results a model for initiation of DNA replication that may be applicable also for other organisms is discussed.